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WO2013033019A1 - Methods for determining the integrity of a biological sample - Google Patents

Methods for determining the integrity of a biological sample Download PDF

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Publication number
WO2013033019A1
WO2013033019A1 PCT/US2012/052519 US2012052519W WO2013033019A1 WO 2013033019 A1 WO2013033019 A1 WO 2013033019A1 US 2012052519 W US2012052519 W US 2012052519W WO 2013033019 A1 WO2013033019 A1 WO 2013033019A1
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WIPO (PCT)
Prior art keywords
sentinel
rna
rnas
degradation
biological sample
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PCT/US2012/052519
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French (fr)
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Curt HAGEDORN
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University Of Utah Research Foundation
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Publication of WO2013033019A1 publication Critical patent/WO2013033019A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • compositions, methods and kits relate broadly to the identification of nucleic acids for determining the overall quality of a biological test sample.
  • the compositions, methods and kits relate to assessing the quantity and processive degradation of nucleic acid molecules as a measure of test sample integrity.
  • RNA sample arrays facilitate the determination of nucleic acid expression levels, which can serve as diagnostic or prognostic indicators of disease.
  • Such tests are clinically employed to aid in the selection of appropriate treatment regimens for patients with various disease indications, such as, e.g., cancer.
  • accurate measures of gene expression can be critical to ensure that the proper therapy is chosen or diagnosis is established.
  • Biological test samples are routinely collected, stored and/or processed under suboptimal conditions for maintaining intact oligonucleotides, e.g., samples may be subjected to room temperature for extended periods of time subsequent to collection.
  • RNases are ubiquitous in biological samples and therefore samples containing RNA are susceptible to degradation. It follows that, if the quality of isolated RNA is compromised, i.e., from a degraded sample, then inaccurate expression profiles may result. Consequently, medical practitioners may prescribe improper treatment protocols or diagnoses based on inaccurate data.
  • compositions, methods and kits for detecting and quantifying nucleic acid markers which serve as a measure of biological sample integrity.
  • the methods described herein measure the quantity and intactness, i.e., level of degradation, of representative RNAs as a conduit for determining the overall quality of a biological test sample.
  • representative RNAs are abundantly expressed and processively degrade with 3 '-5' polarity.
  • methods for determining the integrity of a biological sample include identifying one or more sentinel RNAs in the biological sample, determining the amount of degradation in the one or more sentinel RNAs, comparing the amount of degradation to a reference standard, and correlating the degradation to the integrity of the biological sample.
  • the biological sample includes one or more of blood, plasma, serum, lymph, mucus, sputum, tears, urine, stool, saliva, tissue, hair, animal cells, and plant cells.
  • the one or more sentinel RNAs are mRNA.
  • the one or more sentinel RNAs include one or more of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orf3, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT, SERPINA5, ATP5C1, RPL13A,
  • the amount of degradation includes one or more selected from the group consisting of: 3 '-5' processive degradation, 5 '-3 ' processive degradation, a ratio of 3 '-5' processive degradation to 5 '-3' processive degradation, and a ratio of 5 '-3 ' processive degradation to 3 '-5' processive degradation.
  • the 3 '-5 ' processive degradation is determined from 200 nucleotides from the 3 '-end of the one or more sentinel RNAs.
  • the 5 '-3' processive degradation is determined from 200 nucleotides from the 5 '-end of the one or more sentinel RNAs.
  • measuring is by RNA sequencing.
  • the measuring is by quantitative PCR.
  • the quantitative PCR is performed using primers specific for the one or more sentinel RNAs including one or more of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orf3, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKNIA, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, C
  • a reference standard is included.
  • the reference standard comprises a known amount of sentinel RNA degradation from a control biological sample.
  • the amount of degradation is measured at one or more time points.
  • the indentifying comprises isolating 5 '-capped RNA in the biological sample and determining the level of degradation at one or more time points.
  • a sentinel R A panel is provided.
  • the panel includes SERPINA3, B2M, ENOl, GNB2L1, TMBIM6, PFN1, MYL6, SAA2, JUNB and TIMP1.
  • a method of identifying one or more sentinel RNAs in a biological sample includes isolating RNA from one or more abundantly expressed genes in the biological sample, measuring processive degradation of the isolated RNA at one or more time points; and classifying the one or more abundantly expressed genes as the one or more sentinel RNAs based on the measuring.
  • the isolated RNA is 5 '-capped RNA.
  • the isolated RNA is mRNA.
  • the processive degradation includes one or more of: 3 '-5' processive degradation, 5 '-3' processive degradation, a ratio of 3 '-5' processive degradation to 5 '-3' processive degradation, and a ratio of 5 '-3' processive degradation to 3 '-5 ' processive degradation.
  • the 3 '-5' processive degradation is from 200 nucleotides from the 3'- end of the one or more sentinel RNAs.
  • the 5 '-3' processive degradation is from 200 nucleotides from the 5 '-end of the one or more sentinel RNAs.
  • a control or standard is included.
  • the control comprises RNA.
  • the processive degradation is rapid compared to the processive degradation from a control RNA.
  • the measuring is by RNA sequencing. Additionally or alternatively, in some embodiments, the measuring is by quantitative PCR.
  • the sentinel RNA includes one or more of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1,
  • AHSG AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1,
  • the quantitative PCR is performed using primers specific for one or more sentinel RNAs.
  • the sentinel RNAs include one or more of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orf3, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIFIA, ENOl, UGT2B4, GABARAPLl, KDELR2, RHBDD2, CD63, F9, TRAMl, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT, SERPINA5, ATP5C1, RPLl 3 A, LBP
  • the biological sample includes one or more of blood, plasma, serum, lymph, mucus, sputum, tears, urine, stool, saliva, tissue, hair, animal cells, and plant cells.
  • kits for testing the integrity of a biological sample are provided.
  • the kit includes one or more primers specific for one or more sentinel RNAs, optionally, reagents for qPCR or RNA sequencing; and degradation reference standards.
  • the kit the primers are specific for the one or more sentinel RNAs including one or more of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orO, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT, SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, ALB, CLNS1A, ARF1, ND
  • the kit comprises PCR primers for sentinel RNAs
  • FIGURE 1 is a series of graphs showing RNA integrity analyses from frozen donor liver biospecimens that were incubated at room temperature (RT) at increase time intervals (0, 5, 10 or 15 minutes).
  • RT room temperature
  • RIN RNA integrity Number
  • FIGURE 2 is a graph showing the percent of protein coding mRNAs with a >50% reduction in the total number of exonic sequencing reads or RPKM (reads per kilobase of gene per million reads) as samples (same as in FIGURE 1) were allowed to thaw at room temperature for 5, 10, and 15 minutes. A >50% reduction in sequencing reads was observed in 2.7% of mRNA transcripts by 5 minutes, 14% at 10 minutes, and 76% by 15 minutes.
  • FIGURE 3 is a graph showing the percent of protein coding mRNAs with >50, >75, and >90%> of their 0 minute sequencing reads after 5, 10, and 15 minutes.
  • FIGURE 4A-4D shows Integrated Genome Browser images showing RNA-seq reads of representative rapidly and slowly degrading mRNAs, isolated from flash frozen human liver directly (0 minutes), and after 5, 10, and 15 minutes at room temperature. Each vertical black bar represents the total number of reads for a 36 nucleotide sequence, originated from 5' capped RNA, and aligned with the corresponding genomic region.
  • Rapidly degrading transcripts PFNl and JUNB (Panel A and B), showed a rapid decrease in number of reads, particularly at the 3' end, as time increased.
  • JUNB showed a 33, 60, and 78% reduction in 3' reads after 5, 10 and 15 minutes, respectively.
  • PFN1 showed a 39, 66, and 86% reduction after 5, 10 and 15 minutes).
  • HIST1H1E Panel C
  • MT-BCOl Panel D
  • FIGURE 5 is a graph showing the integrity of protein coding Pol II RNAs as measured by sequencing 200 bp of their 3' and 5' ends.
  • the RNA-seq data from the experiment described in Figure 1 and Materials and Methods was further analyzed with informatics programs.
  • To calculate a 375' ratio for each RNA transcript the number of sequence reads from the last 200 bases (3' end) was divided by the reads from the first 200 bases (5' end) of each mRNA transcript and graphed according to time.
  • Protein coding mRNAs with a minimum 5' end RPKM > 10 at all time points were included in the analysis to avoid confounding the analysis with genes with very low expression levels.
  • FIGURE 6 show pie charts of mappable Pol II RNA sequencing reads in intronic, exonic, and intergenic regions over time. The percentage of sequencing reads mapping to exonic, intronic, and intergenic regions at 0, 5, 10, and 15 minutes are shown schematically. With increasing time fewer reads were aligned to exonic regions, but not to intronic or intergenic RNAs. For each pie chart, the largest section is exons; the second largest section is introns and the smallest is intergenic.
  • FIGURE 7A-7J are graphs showing RNA-sequence and 375' qRT-PCR data of sentinel mRNAs with time.
  • RNA-sequence data for five sentinel mRNAs mined from the database obtained from the biospecimens described in Figure 1 are presented.
  • B2M Panel A, B
  • SERPINA3 Panel C, D
  • GNB2L1 Panel E, F
  • TMBIM6 Panel G, H
  • ENOl Panel I, J
  • the RNA-sequence data is presented as in Figure 4.
  • FIGURE 8A-8E are graphs showing RNA-sequence data of other sentinel mRNAs with time.
  • FIGURE 9A-9B are graphs showing examples of control mRNAs that exhibit little degradation with time.
  • the RNA-sequence reads for MT-C01 (9 A) are shown as in Figure 7 and the 375' qPCR ratio (9B) is shown below.
  • the RNA-sequence reads for HIST1H1E ( Figure 4C) another sequence that shows slow or no degradation, is also shown herein.
  • FIGURE 10A-C are graphs showing 375 ' end ratios of B2M(A), GNB2L 1 (B) and TMB1M6(C) sentinel mRNAs at different time points (0, 5, 10 and 15 minutes) as determined by qPCR. Samples were total RNA that had ribosomal RNA removed. Values represent mean 375' ratios ⁇ SEM of three replicates at each time.
  • sentinel RNAs amplification, and quantification of representative nucleic acids, e.g., sentinel RNAs, which can be used to quickly and accurately measure the usefulness and reliability of a biological sample by linking sentinel RNA integrity, e.g., intactness and/or degradation levels, to biological sample quality.
  • the methods can be performed in a multiplex format which permits the determination of expression levels for two or more sentinel RNAs in a single reaction.
  • oligonucleotide includes a plurality of oligonucleotide molecules, and a reference to "a nucleic acid” is a reference to one or more nucleic acids.
  • a nucleic acid is a reference to one or more nucleic acids.
  • 3'-end or “3'-region” refer to the portion of a polynucleotide or oligonucleotide, e.g., RNA or DNA, located towards the 3'-end of the polynucleotide or oligonucleotide, and may or may not include the 3' most nucleotide(s) or moieties attached to the 3' most nucleotide of the same polynucleotide or oligonucleotide.
  • 5'-end or “5'-region” refer to the portion of a polynucleotide or oligonucleotide, e.g., RNA or DNA, located towards the 5' end of the polynucleotide or oligonucleotide, and may or may not include the 5' most nucleotide(s) or moieties attached to the 5' most nucleotide of the same polynucleotide or oligonucleotide.
  • amplification or “amplifying” refers to the production of additional copies of a nucleic acid sequence. Amplification is typically performed by using, for example, polymerase chain reaction (PCR), reverse transcription RT-PCR, qPCR, qRT- PCR, etc., technologies and/or real time PCR and/or other technologies known in the art.
  • PCR polymerase chain reaction
  • qPCR reverse transcription RT-PCR
  • qRT-PCR qRT-PCR
  • qRT-PCR qRT-PCR
  • qRT-PCR qRT-PCR
  • PCR mixture refers to an aqueous solution comprising the various reagents used to amplify a target nucleic acid, e.g. , RNA, DNA, cDNA, etc., and the like.
  • Amplification may be exponential or linear.
  • a nucleic acid to be amplified may be, for example, either RNA, DNA, cDNA, and the like or equivalents or complements thereof.
  • the sequences amplified in this manner form an "amplicon.” While the exemplary methods described hereinafter relate to amplification using PCR, qPCR, or qRT-PCR, numerous other methods are known in the art for amplification of nucleic acids, e.g., isothermal methods, rolling circle methods, etc.
  • comparing two or more samples means that the same type of sample, e.g., a tissue sample, is used in the comparison.
  • nucleic acid degradation e.g., DNA or RNA
  • comparable samples may be obtained from the same individual at different times.
  • comparable samples may be obtained from different individuals, e.g., a patient and a healthy individual.
  • comparable samples are normalized by a common factor.
  • body fluid samples are typically normalized by volume body fluid and cell-containing samples are normalized by protein content or cell count.
  • determining means determining if a characteristic, trait, or feature is present or not. Assessing may be relative or absolute. Assessing the presence of, for example, includes determining the amount of something present, as well as determining whether it is present or absent.
  • the phrases “difference in the level of or “difference in the amount of refer to differences in the quantity of a particular biomarker, e.g., one or more nucleic acids, e.g., RNAs, in a sample as compared to a control or reference level.
  • the quantity of a particular RNA may be present at an elevated amount or at a decreased amount in samples of patients with a disease compared to a reference level.
  • a "difference in the amount of may be a difference between the level of marker present in a sample as compared to a control.
  • the difference in the amount of is at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%), at least about 25%, at least about 30%>, at least about 35%, at least about 40%>, at least about 50%), at least about 60%>, at least about 75%, at least about 80%> or more.
  • a "difference in the amount of can be a statistically significant difference between the level of the marker present in a sample as compared to a control. For example, a difference can be statistically significant if the measured level of the marker R A falls outside of about 1.0 standard deviations, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 stand deviations of the mean of any control or reference group.
  • RNA expression refers to the process of converting genetic information encoded in a gene into RNA, e.g., total RNA, mRNA, miRNA, rRNA, tRNA, or snRNA, through transcription of the gene, i.e., via the enzymatic action of an RNA polymerase, and for protein encoding genes, into protein through translation of mRNA.
  • Gene expression can be regulated at many stages in the process. Up- regulation or activation refers to regulation that increases the production of gene expression products, i.e., RNA or protein, while down-regulation, repression or knock-down refers to regulation that decrease production.
  • Molecules e.g., transcription factors that are involved in up-regulation or down-regulation are often called activators and repressors, respectively.
  • nucleic acid refers to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof and to naturally occurring or synthetic molecules. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, or to any DNA-like or RNA-like material, including natural and/or non-natural bases.
  • microarray refers to an arrangement of a collection of nucleic acids, e.g., nucleotide sequences in a centralized location.
  • Arrays can be on a solid substrate, such as a glass slide, or on a semi-solid substrate, such as nitrocellulose membrane.
  • the nucleotide sequences can be DNA, RNA, or any combination or permutations thereof.
  • the nucleotide sequences can also be partial sequences or fragments from a gene, primers, whole gene sequences, non-coding sequences, coding sequences, published sequences, known sequences, or novel sequences.
  • Tissue microarrays are well known in the art and can be performed as described. See e.g., Camp, R. L., et al, J Clin Oncol, 26, 5630-5637 (2008).
  • a "primer” for amplification is an oligonucleotide that specifically anneals to a target or marker nucleotide sequence and forms a substrate for a nucleic acid polymerase.
  • primer includes "primer pairs" required to amplify a nucleic acid sequence.
  • the 3' nucleotide of the primer can be complementary, identical, and/or hybridize to a target or marker sequence at a corresponding nucleotide position for optimal primer extension by a polymerase.
  • a "forward primer” is a primer that anneals to the anti-sense strand of double stranded DNA (dsDNA) or an appropriate cDNA or RNA or other nucleic acid with similar polarity.
  • a “reverse primer” anneals to the sense- strand of dsDNA or an appropriate cDNA or RNA or other nucleic acids with similar polarity.
  • prognosis refers to a prediction of the probable course and outcome of a clinical condition or disease.
  • a prognosis is usually made by evaluating factors or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease.
  • determining the prognosis refers to the process by which the skilled artisan can predict the course or outcome of a condition in a patient.
  • prognosis does not refer to the ability to predict the course or outcome of a condition with 100% accuracy.
  • prognosis refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition.
  • prognosis and positive prognosis or “unfavorable prognosis” and “negative prognosis” as used herein are relative terms for the prediction of the probable course and/or likely outcome of a condition or a disease. A favorable or positive prognosis predicts a better outcome for a condition than an unfavorable or negative prognosis.
  • a "favorable prognosis" is an outcome that is relatively better than many other possible prognoses that could be associated with a particular condition, whereas an unfavorable prognosis predicts an outcome that is relatively worse than many other possible prognoses that could be associated with a particular condition.
  • Typical examples of a favorable or positive prognosis include a better than average cure rate, a lower propensity for metastasis, a longer than expected life expectancy, differentiation of a benign process from a cancerous process, and the like.
  • a positive prognosis is one where a patient has a 50% probability of being cured of a particular disease, e.g., cancer, after treatment, while the average patient with the same cancer has only a 25% probability of being cured.
  • a reference level refers to a sample having a level or amount of a substance which may be of interest for comparative purposes.
  • a reference level is the average of the amount of, and/or rate of, intact and/or degraded nucleic acid (e.g., sentinel RNA) from one or more biological samples taken from a control population of one or more healthy (disease-free) subjects.
  • nucleic acid e.g., sentinel RNA
  • the reference level is the amount of, and/or rate of, intact and/or degraded nucleic acid from the same subject at a different time, e.g., prior to the subject developing the disease and/or prior to and/or after and/or during therapy, and/or before and after a sample preparation procedure, and/or before and after sample storage.
  • samples are normalized by a common factor.
  • body fluid samples are normalized by volume body fluid and cell-containing samples are normalized by protein content or cell count.
  • sample biological sample
  • test sample test sample
  • clinical sample laboratory sample
  • biological sample biological sample
  • test sample test sample
  • clinical sample laboratory sample
  • biological sample biological sample
  • biological sample biological sample
  • test sample test sample
  • clinical sample laboratory sample
  • laboratory sample and/or “biospecimen” are used interchangeably and in the broadest sense.
  • a sample may include a bodily tissue or a bodily fluid including but not limited to tissue samples, blood (or a fraction of blood such as plasma or serum), lymph, mucus, tears, urine, stool and saliva.
  • a sample may include an extract from an animal or plant cell, a chromosome, organelle, or a virus.
  • a sample may be a "cell-free” sample, meaning that the volume of cells in the sample are less than about 2% of the total sample volume (preferably less than about 1% of the total sample volume).
  • a sample may comprise RNA, e.g., mRNA or cDNA, any of which may be amplified to provide amplified nucleic acid.
  • a sample may include nucleic acid in solution or bound to a substrate, e.g. , as part of a microarray.
  • a sample may be obtained from any subject or any patient.
  • sample integrity or “sample quality” or “overall quality” are used interchangeably and refer the overall “health” of a sample as it relates to the amount, level, intactness, and/or rate of nucleic acid degradation, e.g., RNA degradation, therein.
  • the “sample integrity” or “sample quality” or “overall quality” can also refer to the ability of the sample to accurately and/or reliably reflect the results for a given assay. Sample integrity or quality, however, is independent of the accuracy or reliability of any particular assay.
  • a biological sample when employing a gene expression panel, may have "optimal” or “good” quality or integrity if the sample possesses the nucleic acids which may be expressed and/or are expressed, e.g., one or more specific mR As.
  • a biological sample when employing a gene expression panel, may have
  • the term "subject” and "patient” are used interchangeably and refer to a mammal, such as a human, but can also be another animal such as a domestic animal, e.g., a dog, cat, or the like, a farm animal, e.g., a cow, a sheep, a pig, a horse, or the like, or a laboratory animal, e.g., a monkey, a rat, a mouse, a rabbit, a guinea pig, or the like.
  • a domestic animal e.g., a dog, cat, or the like
  • a farm animal e.g., a cow, a sheep, a pig, a horse, or the like
  • laboratory animal e.g., a monkey, a rat, a mouse, a rabbit, a guinea pig, or the like.
  • sentinel RNA or “sentinel RNAs” refer to one or more RNA molecules capable of serving as a biomarker for the quality of RNA, such as mRNA, in a biological sample.
  • the sentinel RNA serves as a biomarker for the quality of RNA, such as mRNA, in a biological sample at a given time point.
  • RNA quality in a biological sample See, e.g., Schroeder et al, "The RIN: an RNA integrity number for assigning integrity values to RNA measurements.” BMC molecular biology, 7, 3 (2006).
  • RNA integrity number (RIN).
  • Pol II RNA polymerase II
  • a RIN of > 7 is considered to be “intact” RNA.
  • Auer et al. “Chipping away at the chip bias: RNA degradation in microarray analysis.” Nature genetics, 35, 292-293 (2003).
  • RIN values of less than 7 may however produce spurious microarray results due to reduced transcript probe length and decreased yields prior to hybridization.
  • Thompson et al. "Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA.” BMC biotechnology, 7, 57 (2007).
  • RNA transcripts possessing RIN scores of greater than 7.8 can be degraded prior to use in hybridization assays. Popova et al, "Effect of RNA quality on transcript intensity levels in microarray analysis of human post-mortem brain tissues.” BMC genomics, 9, 91 (2008).
  • RNA sequencing RNA-seq
  • 5'-methyl guanosine capped 5'-capped or 5'-m 7 GpppN
  • the degree of degradation in a biological sample can be quantitatively determined using techniques known in the art, e.g., PCR, RT-PCR, and/or qRT-PCR, while avoiding the inconsistencies associated with 3 '-end RNA isolation.
  • PCR PCR, RT-PCR, and/or qRT-PCR
  • sentinel RNA identification of 5'-m 7 GpppN capped, abundantly expressed and rapidly degrading RNAs, termed sentinel RNA, which can be used to accurately measure the quality or integrity of a biological sample.
  • sentinel RNA degradation and the concomitant level of intactness, are assessed to identify abundantly expressed genes, which degrade at a rapid rate, thereby allowing for the absolute and/or relative determination of 3'- and/or 5'-end decay of these candidate RNAs.
  • determinations provide sentinel RNAs as a tool for assessing the overall quality of a biological sample, prior to, for example, clinical assessment and/or gene array analysis.
  • sentinel RNA refers to one or more RNA molecules capable of serving as a biomarker for the quality of RNA, such as mRNA, in a biological sample.
  • sentinel RNA serve as biomarkers for the quality of RNA, such as mRNA, in a biological sample at a given time point.
  • sentinel RNA imparts a mechanism for determining the integrity or quality of a biological sample.
  • sentinel RNAs are abundantly expressed, e.g.
  • tissues and cell types such as, but not limited to, e.g., one or more of muscle cells or tissue, epithelial cells or tissue, endothelia cells or tissue, organ cells or tissue, stem cells or tissue, umbilical vessel cells or tissue, corneal cells or tissue, cardiomyocytes, aortic cells or tissue, corneal epithelial cells or tissue, aortic endothelial cells or tissue, fibroblasts, hair cells or tissue, keratinocytes, melanocytes, adipose cells or tissue, bone cells or tissue, osteoblasts, airway cells or tissue, microvascular cells or tissue, mammary cells or tissue, vascular cells or tissue,
  • chondrocytes placental cells or tissue, plant cells, and the like.
  • one or more sentinel RNAs are tissue specific. Additionally or alternatively, in some embodiments
  • one or more sentinel RNAs do not possess a specific tropism.
  • sentinel RNAs are typically degraded at a rapid, or comparatively measurable, rate, e.g., not degradation resistant. Therefore, sentinel RNAs include, but are not limited to, biologically active or expressed RNA molecules, such as, e.g., exonic RNA (exons), messenger RNA (mRNA), microRNA (miRNA), transfer RNA (tRNA), and/or any RNA molecule possessing a 5'-m 7 GpppN cap.
  • exonic RNA exonic RNA
  • mRNA messenger RNA
  • miRNA microRNA
  • tRNA transfer RNA
  • sentinel RNAs include, for example, biologically inert RNAs, such as, e.g., intronic RNA (introns), intergenic RNA, and the like, or any RNA species derived from the aforementioned classes of RNAs by metabolic processes.
  • biologically inert RNAs such as, e.g., intronic RNA (introns), intergenic RNA, and the like, or any RNA species derived from the aforementioned classes of RNAs by metabolic processes.
  • sentinel RNAs include, but are not limited to, e.g., RNAs from one or more of the following genes or gene products: EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orO, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT, SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, A
  • the one or more sentinel RNAs are one or both of GNB2L1 and TMBIM6.
  • the sentinel RNAs of the present technology can be directly or indirectly assayed.
  • one or more sentinel RNAs are used as a template for quantitative amplification, e.g., qRT-PCR, according to the methods disclosed herein.
  • the sentinel RNAs described herein, as listed above can be grouped in non-limiting assay panels for use in the methods described herein. In some embodiments, at least from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or 500 sentinel RNAs to about 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 1,000, or 10,000 sentinel RNAs are group into arrays in accordance with the methods provided herein. Additionally or alternatively, in some embodiments, the one or more sentinel RNAs are assayed in a multiplexed format.
  • compositions, methods and kits described herein may be used to detect nucleic acids associated with various genes using a biological sample obtained from a subject.
  • the biological sample may be from any organism that possesses endogenous nucleic acid (RNA and/or DNA).
  • Biological samples can be obtained by standard procedures and can be used immediately or stored, under conditions appropriate for the type of biological sample, for later use. Methods of obtaining biological samples are well known to those of skill in the art and include, but are not limited to, aspirations, tissue sections, swabs, drawing of blood or other fluids, surgical or needle biopsies, and the like. Additionally or alternatively, in some embodiments, biological samples are obtained pursuant to the instructions or recommendations of a bioassay manufacturer (e.g., MammaPrintTM, ColoPrintTM, etc.) with which the sample is to be tested.
  • a bioassay manufacturer e.g., MammaPrintTM, ColoPrintTM, etc.
  • the starting material for the assays disclosed herein typically include, but are not limited to, one or more clinical samples, which are suspected to contain RNA, e.g., total cellular RNA, and/or one or more sentinel RNAs.
  • the sample may be obtained from a subject or patient.
  • the biological sample in some embodiments, is a cell-containing liquid or a tissue. Samples may include, but are not limited to, biopsies, blood, blood cells, bone marrow, fine needle biopsy samples, peritoneal fluid, amniotic fluid, plasma, pleural fluid, saliva, semen, serum, tissue or tissue homogenates, frozen or paraffin sections of tissue.
  • Samples may also be processed, such as sectioning of tissues, fractionation, purification, or cellular organelle separation.
  • the biological sample includes one or more of blood, plasma, serum, lymph, mucus, sputum, tears, urine, stool, saliva, tissue, hair, animal cells, and plant cells.
  • the sample may contain cells, tissues or fluid obtained from a patient suspected being afflicted with a disease such as, but not limited to, e.g., exogenous diseases, including, but not limited to, bacterial, fungal, prion, and/or viral diseases, e.g., chronic viral hepatitis, and/or endogenous diseases, such as, but not limited to, cancers including, but not limited to, e.g., lymphatic cancers, hematologic cancers, breast cancer, liver cancer, lung cancer, prostate cancer, gastric cancer, endometrial cancer, salivary gland cancer, adrenal cancer, non-small cell lung cancer, pancreatic cancer, renal cancer, ovarian cancer, peritoneal cancer, head and neck cancer, bladder cancer, colorectal cancer, glioblastomas, hematologic tumors, multiple myeloma, acute myelogenous leukemia, and/or colon cancer, and/or metastatic cancers of the same.
  • exogenous diseases including, but not limited to
  • Methods for isolating a particular cell from other cells in a sample include, but are not limited to, Fluorescent Activated Cell Sorting (FACS) as described, for example, in Shapiro, Practical Flow Cytometry, 3rd edition Wiley-Liss; (1995), density gradient centrifugation, or manual separation using micromanipulation methods with microscope assistance.
  • FACS Fluorescent Activated Cell Sorting
  • Exemplary cell separation devices that are useful in the invention include, without limitation, a Beckman JE-6 centrifugal elutriation system, Beckman Coulter EPICS ALTRA computer-controlled Flow Cytometer-cell sorter, Modular Flow Cytometer from Cytomation, Inc., Coulter counter and channelyzer system, density gradient apparatus, cytocentrifuge, Beckman J-6 centrifuge, EPICS V dual laser cell sorter, or EPICS PROFILE flow cytometer.
  • a tissue or population of cells can also be removed by surgical techniques.
  • a biological sample can be prepared for use in the methods of the present invention by lysing a cell that contains one or more desired nucleic acids, e.g., total RNA, mRNA, 5 '-capped RNA, and/or sentinel RNAs.
  • a cell is lysed under conditions that substantially preserve the integrity of the desired nucleic acid.
  • cells can be lysed or subtractions obtained under conditions which stabilize RNA and/or DNA.
  • Such conditions include, for example, cell lysis in strong denaturants, including chaotropic salts such as guanidine thiocyanate, ionic detergents such as sodium dodecyl sulfate, organic solvents such as phenol, high lithium chloride concentrations or other conditions known in the art to be effective in limiting the activity of endogenous RNases during RNA purification.
  • chaotropic salts such as guanidine thiocyanate
  • ionic detergents such as sodium dodecyl sulfate
  • organic solvents such as phenol
  • high lithium chloride concentrations such as RNA purification
  • relatively undamaged nucleic acids such as RNA can be obtained from a cell lysed by an enzyme that degrades the cell wall.
  • Cells lacking a cell wall either naturally or due to enzymatic removal can also be lysed by exposure to osmotic stress.
  • Other conditions that can be used to lyse a cell include exposure to detergents, mechanical disruption,
  • the nucleic acids are separated from proteins and sugars present in the original sample and/or a sample that was subjected to cell lysis. Any purification methods known in the art may be used in the context of the present invention. Those skilled in the art will know or be able to readily determine methods for isolating nucleic acid from a cell, fluid or tissue, such as those described in Sambrook et ah,
  • Nucleic acid sequences in the sample can subsequently be amplified using in vitro amplification, such as PCR or qPCR. Typically, compounds that may inhibit polymerases are removed from the nucleic acids.
  • the biological samples ⁇ e.g., cells, tissues, fluids, nucleic acid, etc. for testing
  • a bioassay manufacturer e.g., MammaPrintTM, ColoPrintTM, etc.
  • RNA isolation and/or extraction is required for a variety of biological assays.
  • Gene expression arrays, RNA sequencing technologies, and tiling arrays, for example, are fundamental tools for developing diagnostic biomarkers of disease, which may require RNA purification. Time consuming annotation of expressed genes and RNA quality assurance in patient samples, however, underlie the problems associated with these approaches.
  • transcript signal values are calculated from probes annealed within 500 bases of the 5'- and/or 3'-end of genes such as, e.g., GAPDH and ACTB.
  • the quality of mRNA is measured as a function of the 375' ratio, wherein quotients of approximately three denote equal amounts of cDNA synthesis from the 5'- and 3 '-ends.
  • Such methods nevertheless typically isolate RNA transcripts from the 3 '-end, via oligo(dT) primers, which hybridize to the poly(A) terminus.
  • 5 '-end RNA levels can be underestimated due to 3 '-end degradation and/or the inability of a reverse transcriptase to processively reach the 5 '-end, i.e., when cDNA synthesis is required.
  • RNA Pol II transcription products which possess a 5'-m 7 GpppN cap (5 '-cap).
  • RNAs include, but are not limited to, for example, exonic mRNA (exons), protein coding mRNA, micro-RNA (miRNA) precursors, and other RNAs which may not encode for specific proteins. See Cai et al, "Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs.” RNA, 10, 1957- 1966 (2004).
  • the isolation and analysis of 5'-capped RNA enables the accurate identification and evaluation of sentinel RNAs, their intactness and/or the 3 -5' and 5 '-3' degradation that occurs in Pol II transcripts because such methods obviate the inconsistencies associated with 3' -RNA isolation.
  • RNA transcripts are present in eukaryotic cells than are currently annotated, yet many appear to lack 3'-poly(A) ends and a majority of these RNAs are Pol II transcripts. See Durtrow et al. (2008). These RNAs likely encode proteins and may also represent non-coding regulatory RNAs.
  • the present technology therefore provides an efficient method of isolating Pol II transcripts by binding their 5 '-caps with a RNA cap binding protein and/or a high-affinity variant of the RNA cap binding protein (4EK119A).
  • RNA sequences in the test sample can subsequently be amplified using in vitro amplification, such as, for example, qRT-PCR.
  • substances, enzymes, and/or compounds that may degrade RNA e.g., RNases, are removed from the sample prior to and/or during RNA isolation and/or extraction.
  • total RNA, mRNA, sentinel RNA, tRNA, rRNA, miRNA, and/or other RNA molecules which may be contained in a biological sample, are isolated.
  • total RNA in some embodiments, is first isolated from a biological sample using techniques known in the art, such as, e.g., Trizol® (Invitrogen), Ribominus®, or other RNA isolation/extraction kits known in the art.
  • Trizol® Invitrogen
  • Ribominus® Ribominus®
  • 5'-capped RNA including, e.g., mRNA and/or sentinel RNA is separated from the total RNA thereby enriching for the mRNA and/or sentinel RNA, for example.
  • the 5 '-capped RNA is isolated from a sample by incubating a tagged cap- binding protein, e.g., eIF-4E, with the total RNA and thereafter purifying the bound complex using methods known in the art, e.g., magnetic or affinity bead isolation and/or chromatography. See, e.g., Folkers et al, "ENCODE tiling array analysis identifies differentially expressed annotated and novel 5 '-capped RNAs in hepatitis C infected liver.” PLoS One, 6, el 4697 (2011). Additionally or alternatively, in some embodiments, a high affinity variant of the cap binding protein is employed to enrich for 5 '-cap-isolated RNA. See Choi et al. (2003).
  • RNA can be isolated from other RNAs in the biological sample to enrich for mRNA and/or sentinel RNA, such that the mRNA and/or sentinel RNA is substantially pure, meaning it is at least about 70%, 75%, 80%, 85%, 90%, 95% pure or more, but typically less than 100%) pure, with respect to other RNA molecules.
  • Various kits for the extraction and/or full or partial isolation and/or purification of total RNA, mRNA, and/or one or more sentinel RNAs, from biological samples are provided herein and/or commercially available and are well known to the skilled artisan.
  • exosome-mediated decay represents one of the primary pathways for mRNA degradation in biological cells. See, e.g., Chen et al., "AU binding proteins recruit the exosome to degrade ARE-containing mRNAs.” Cell, 107, 451-464 (2001). Exosome-mediated decay occurs by 3 -5'
  • AREs AU-rich elements
  • sentinel RNAs are identified by (A) isolating 5' capped mRNA (e.g., as described above in section III.B), and (B) analyzing these RNAs to determine sequence identity, abundance, and/or sequence integrity (e.g., degradation patterns).
  • candidate sentinel RNAs can be identified by nucleic acid sequencing, in conjunction with bioinforomatics programs and publicly available gene expression databases, such as, e.g., GEO Dataset-GSE5364, which contain genetic profiles from a variety of diseased and corresponding healthy samples, and/or databases such as, for example, the Ensembl human genome database, to identify the candidate sentinel RNA sequences.
  • bioinformatics programs can be used to determine the relative abundance and/or integrity (e.g., degradation patterns) of the candidate sentinel RNAs.
  • RNA and/or DNA sequencing allows for the identification of candidate sentinel RNAs the subsequent evaluation of abundance and degradation patterns of these RNAs.
  • RNA and/or DNA sequencing can be performed using methods known in the art, such as, for example, dideoxy chain termination method of Sanger et al. , Proceedings of the National Academy of Sciences USA, 74, 5463-5467 (1977), with modifications by Zimmermann et al, Nucleic Acids Res., 18: 1067 (1990). Sequencing by dideoxy chain termination method can be performed using Thermo Sequenase (Amersham Pharmacia, Piscataway, NJ), Sequenase reagents from US Biochemicals or Sequatherm sequencing kit (Epicenter Technologies, Madison, Wis.). Sequencing may also be carried out by the "RR
  • dRhodamine Terminator Cycle Sequencing Kit from PE Applied Biosystems (product no. 403044, Rothstadt, Germany), Taq DyeDeoxyTM Terminator Cycle Sequencing kit and method (Perkin-Elmer/ Applied Biosystems) in two directions using an Applied Biosystems Model 373 A DNA or in the presence of dye terminators CEQTM Dye Terminator Cycle Sequencing Kit, (Beckman 608000). Additionally or alternatively, sequencing can be performed by a method known as Pyrosequencing (Pyrosequencing, Westborough, Mass.). Detailed protocols for Pyrosequencing can be found in: Alderborn et al., Genome Res. (2000), 10: 1249-1265.
  • R A or DNA sequencing can be performed using hybridization techniques, such as, but not limited to, heteroduplex tracking assays, line probe assays, nucleic acid arrays, nucleic acid arrays (DNA chips), bead arrays, and the like. See U.S. Pat. No. 6,300,063 and U.S. Pat. No. 5,837,832.
  • Massively parallel sequencing, next generation sequencing, and/or deep sequencing are also be used in the methods described herein. See Voelkerding et al. (2009) Clin. Chem. 55(4):641-658; ten Bosch et al. (2008) J. Mol. Diag. 10(6):484-492;
  • Genome Sequence FLX systems from 454 Corporation (a Roche Company)
  • Genome Analyzer from Illumina
  • SOLiD system from Life Technologies.
  • Single molecule massively parallel sequencers include those from, for example, Helicos Biosciences (the Heliscope) and Pacific Biosciences.
  • next generation RNA sequencing is employed to measure the amount, level, and/or presence of one or more candidate sentinel RNAs.
  • Pol II RNA transcripts are analyzed in some embodiments by RNA sequencing using, e.g., an Illumina Genome Analyzer 2 (GA2) and standard protocols for preparing and sequencing libraries representing RNA samples, as known in the art.
  • G2 Illumina Genome Analyzer 2
  • 5'-capped RNAs and/or total RNA is sequenced. Total RNA and/or 5 '-capped RNA- with rRNA removed in some embodiments-can be sequenced using the Ribominus® for RNA-seq kit (Invitrogen).
  • RNAs are fragmented, reverse transcribed to cDNA, and adapted to create a cDNA library.
  • Adapters for cDNAs can be amplified via PCR using, e.g., a Cluster Station (Illumina) and sequenced with an Illumina GA2. See Oler et al., "Human RNA polymerase III transcriptomes and relationships to Pol II promoter chromatin and enhancer-binding factors.” Nat Struct Mol Biol, 17, 620-628 (2010).
  • Nucleic acid sequencing allows for the identification of the candidate sentinel RNA, and provides the information necessary to develop amplification primers which can be used in the methods discussed below.
  • candidate sentinel RNA sequences are aligned with sequences from one or more databases, such as, but not limited to, for example, the NCBI human genome database, NCBI 37.3 of the human genome ⁇ e.g., using the Bowtie aligner), the UCSC Genome Browser, MXSCARNA, Murlet, RNAmine, SCARNA, PHMMTS, PSTAG, Rfold, Stem Kernels, CentroidAlifold, CentroidHomfold, CentroidAlign, miRRim, SCARNA LM, npbfold, RactIP, IPknot, Raccess, Fdur, Rentropy, Rchange GEO Datasets, and/or the Ensembl human genome database, and the like.
  • bioinformatic programs can then be employed to identify the candidate sentinel RNA sequences, and/
  • RNA sequence Reads Per Kilobase of gene per Million reads (“RPKM” or "sequence reads" are obtained and/or performed. Additionally or alternatively, in some embodiments, from about 1,000,000 to about from 10,000,000 RPKM are obtained and/or performed. Additionally or alternatively, in some embodiments 1, 2, 5, 10, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 RPKM are obtained and/or performed.
  • RPKM from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or 40 nucleotides to about 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, or 500 nucleotides are aligned to, for example, NCBI 37.3 of the human genome, using the Bowtie aligner. See Oler et al., "Human RNA polymerase III transcriptomes and relationships to Pol II promoter chromatin and enhancer-binding factors.” Nat. Struct. Mol. Biol, 17, 620-628 (2010). Additionally or alternatively, in some embodiments, about 30-50 nucleotides are aligned. Id.
  • Alignments can be filtered and selected based on score ranges for example, based on the number of base mismatches (e.g., between 0 and 3 mismatches).
  • Non-limiting applications such as, for example, "USeqDefmedRegionScanSeqs" can be employed to score genes for relative differential expression.
  • Useq DefinedRegionsScanSeqs determines the genie sequencing reads of a given gene by summing the reads originating from each exon of that gene.
  • DRSS uses the genomic coordinates for each of those three exons to calculate the number of reads from each exon region and sums those reads for that gene. See also, e.g., Nix et ah, "Empirical methods for controlling false positives and estimating confidence in ChlP-Seq peaks.” BMC Bioinformatics, 9, 523 (2008).
  • abundant expression is determined using one or more publicly available gene expression datasets ⁇ e.g., GEO Dataset, containing gene profiles from many solid cancers, GSE5364, Ensembl, etc.) and/or other bioinformatic programs as known in the art.
  • identified sequences ⁇ e.g., candidate sentinel RNAs
  • background levels for read counts of RNA sequencing data are considered to fall between 0.1 and 1 RPKM. The deeper the sequencing depth the more confidence exists in lower background level cutoffs. An RPKM between 0.3-0.4 has been published recently ⁇ see e.g., Blood.
  • abundant expression is defined as three, four, five, six, seven, eight, nine or ten orders of magnitude above background.
  • candidate sentinel RNAs are selected from all genes in the Ensembl human genome.
  • criteria used to identify candidate sentinel RNAs include, for example, expressed genes with about 1, 5, 10, 20, 30, 40, 50, 100, 500, 1000 or more RPKMs. Additionally or alternatively, in some embodiments, candidate sentinel RNAs are selected from samples containing expressed genes with an RPKMs of about > 10. Additionally or alternatively, in some embodiments, candidate sentinel RNAs are selected from samples containing expressed genes with a RPKM of one log above background.
  • sentinel RNAs can include, but are not limited to abundantly expressed genes with about 1 , 5, 10, 20, 30, 40, 50, 100, 500, 1000 or more RPKMs from the 5'- and/or 3'-terminal ends. Additionally or alternatively, in some embodiments, sentinel RNAs contain abundantly expressed genes with about 10-50 or more RPKMs in the 5'- and/or 3'-terminal ends.
  • sequencing analyses of sentinel RNAs are employed to interrogate the 5' and 3'-ends, separately, simultaneously, or sequentially.
  • from about 10, 20, 30, 40, 50, 100, 200, 300, 500, 1000, or 10,000 bases to about 50, 100, 200, 300, 500, 1 ,000; 10,000; or 100,000 bases from the 5*- and/or 3*-ends of one or more sentinel RNAs are analyzed.
  • from about 100 to about 300 bases from the 5' and/or 3'-ends of one or more sentinel RNAs are analyzed.
  • about 200 bases from the 5' and/or 3'-ends of one or more sentinel RNAs are analyzed.
  • sentinel RNAs possess decreasing or increasing 375' and/or 573 ' ratios-at increasing time intervals-relative to static RNAs and/or RNAs which do not possess such ratios. Additionally or alternatively, in some embodiments, sentinel RNAs possess decreasing 375' ratios as time increases.
  • sentinel RNAs processively decay with 3'-5' and/or 5 '-3 ' polarity. Additionally or alternatively, in some embodiments, candidate sentinel RNAs processively decay with 3 '-5' polarity. Additionally or alternatively, in some embodiments, sentinel RNAs processively decay with 3'-5' polarity at a rapid rate.
  • a rapid decay rate is considered a > 20% decrease in sequencing reads on the 3 ' end (200 terminal nucleotides of the 3 ' untranslated end or UTR) compared to the 5 ' end of sentinel RNAs after 5 and 10 minutes at degradation (room temperature in the examples provided). Additionally or alternatively, in some embodiments, a rapid decay rate is defined as a slope of 0.2 between 0 and 10 minutes of degradation.
  • RPKM measurements for identifying sentinel RNAs are selected from sequencing reactions performed after 1 , 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 75, 100, 200, 500, 1000 or more minutes (min) of sample incubation. Additionally or alternatively, in some embodiments, RPKM measurements for identifying sentinel RNAs are selected from sequencing reactions performed after 5, 10, and/or 15 minutes of sample incubation times.
  • the samples are incubated at about from 0°C, 4°C, 5°C, 10°C, 15°C, 20°C, 25°C, and/or 37°C to about from 4°C, 5°C, 10°C, 15°C, 20°C, 25°C, 35°C, 50°C and/or 100°C and evaluated at specific time points (e.g., such as those described above).
  • the samples are incubated at room temperature and evaluated at specific time points (e.g., such as those described above).
  • sentinel RNAs are identified via degradation pattern analysis of candidate sentinel RNAs.
  • 5' capped RNA sequences are compared to sequences provided in one or more database(s), using e.g., bioinformatics programs, to identify those RNAs which are (1) abundantly expressed and (2) show rapid degradation relative to the other 5' capped RNAs.
  • candidate sentinel RNA degradation patterns are determined by calculating the ratio of the number of sequence reads in a defined number of 3' and/or 5 '-end nucleotides. Additionally or alternatively, in some embodiments, the number of sequence reads in a defined number of 3' and/or 5' end nucleotides is determined at different time points to establish a 3 ' end/5 '-end ratio.
  • the defined number of nucleotides in the 3' and 5 '-ends can be the same or different.
  • the defined number of nucleotides ranges from about 5-1000, about 10-500, about 20-250, and about 30-100.
  • the defined number of nucleotides is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or is about 300 or more.
  • the defined number of nucleotides of the 3' and/or 5'-end is 200.
  • candidate sentinel RNA decay is measured at different time points, e.g., from the time a sample is collected and/or different time points from when a sample is thawed.
  • samples are evaluated for degradation at from about 0.1, 0.5, 1, 2, 3, 4, 5 or 6 seconds, minutes, hours, days or years to about from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50 or 100 seconds, minutes, hours, days or years.
  • sentinel RNA decay is measured at about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 30 minutes, 60 minutes, or 90 minutes or more after sample collection or after sample thaw. Additionally or alternatively, in some embodiments, sentinel RNA decay is measured at time points which simulate sample treatment prior to a biological assay. [0093] In a non-limiting exemplary embodiment, candidate sentinel RNAs are identified as follows. Biological samples continaing RNA are sequenced at TO, Tl, T2, and T3 of incubation at a given temperature. Sequencing is performed such that each sample has given number of sequence reads (e.g., 1-10 million sequence reads) for a fixed number of nucleotides (e.g., 10-50) nucleotides. Sequences are evaluated as follows.
  • RNAs with an overall decrease in 375' ratios are identified: (e.g., those RNAs with a slope for the best fit linear line of ⁇ -0.2).
  • RNAs with progressive 3' to 5' degradation are identified (e.g., those RNAs that show a difference in 375' ratio of > 0.2 between TO, Tl and T2 time points).
  • the amount or level of one or more sentinel RNAs present in a biological sample correlates with the biological integrity of the sample, which may subsequently be used in a diagnostic assay. Accordingly, in some aspects, the present methods can be used to measure sentinel RNA levels or amounts in order to facilitate disease diagnosis and/or therapeutic administration.
  • the methods provided herein can be applied to quantify the amount of sentinel RNA decay, and concomitant intactness, in a biological sample at one or more time points.
  • Embodiments of the present technology include methods for determining the amount of one or more sentinel RNA sequences present in a biological sample, and/or degradation thereof, and comparing the result to a reference standard, such as, e.g., a standard curve, normal or healthy control samples, etc.
  • a reference standard such as, e.g., a standard curve, normal or healthy control samples, etc.
  • the difference in the level or amount of one or more sentinel RNA sequences in the biological sample, compared to the reference standard is indicative of the biological sample quality, which may be
  • sentinel R A levels are determined in a biological sample by amplification.
  • the sentinel R As may be biomarkers which pertain to a subject and/or patient and can be used to measure the quality or integrity of a sample, and/or the progressive decrease in the quality or integrity of a biological sample as a function of time.
  • sentinel RNA standards are developed to simulate sample treatment prior to a biological assay (e.g., a MammaPrintTM Oncotype DXTM or ColoPrintTM assay).
  • Standards can be prepared from a separate, comparable biological sample or the same biological sample to be assayed.
  • standards can be prepared and evaluated in advance of the biological assay, using an aliquot of the biological sample to be assayed or a comparable sample.
  • controls or standards can be performed and control or standard information can be provided as values or ranges that indicate sample integrity, to be compared to the sentinel RNA degradation value or range of the biological sample.
  • the biological sample integrity is suspect and should not be used in the bioassay, or if used in the assay, results derived from the suspect biological sample should be critically analyzed.
  • the biological sample shows sentinel RNA degradation that falls within an "acceptable" range or value indicated by the reference standard, the biological sample integrity is good, and the bioassay should proceed.
  • RNA isolation or purification RNA isolation or purification
  • sample storage steps e.g., sample storage steps, and/or the final preparation steps of the sample prior to use in the selected assay application (e.g., reagent addition, incubation, etc.).
  • time, temperature, reagent addition and/or storage of the sample are considered in developing a reference sample(s).
  • sentinel RNA decay is measured to develop a standard from about 0.1, 0.5, 1, 2, 3, 4, 5 or 6 seconds, minutes, hours, days or years to about from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50 or 100 seconds, minutes, hours, days or years after sample collection and/or after sample thaw (e.g., at RT, or at 4°C).
  • sentinel RNA decay is measured after RNA is isolated from the standard sample, to develop the standard. Additionally or alternatively, in some embodiments, RNA is isolated by methods known in the art, including but not limited to, for example, Trizol® kit and/or 5 '-capped mRNA isolation.
  • the integrity of a biological sample is compared to standard samples containing one or more sentinel RNAs which have been incubated at a given temperature or temperature range (e.g., 0°C, 4°C, 25°C, room temperature (RT), 37°C, etc.). Additionally or alternatively, the incubation of the control sample is at a temperature from about 1, 10, 20, 30 or 40°C to about 20, 30, 40, 50, 60, 70, 80, 90, 100°C or higher. Additionally or alternatively, a standard curve can be generated to facilitate accurate comparative analyses.
  • the integrity of a biological sample can be compared to standard samples containing one or more sentinel RNAs which have been incubated at a selected temperature from about 0.1, 0.5, 1, 2, 3, 4, 5 or 6 seconds, minutes, hours, days or years to about from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50 or 100 seconds, minutes, hours, days or years.
  • sentinel RNA decay to develop standards is measured using the same methods as used for determining sentinel RNA decay in the biological sample.
  • one or more standard curves can be developed and used as a standards or controls.
  • a control sample is collected and subject to RNase treatment, and/or incubation at a particular temperature, with aliquots being evaluated for sentinel RNA degradation at pre-determined time points (e.g., once per 30 seconds, once per minute, every 2 minutes, every 5 minutes, etc.).
  • a control sample is collected and subject to RNase treatment at increasing concentrations of RNase, and/or incubation at a particular temperature and/or for a specific time (e.g., once per 30 seconds, once per minute, every 2 minutes, every 5 minutes, etc.).
  • control or standard values of sentinel RNA decay are measured using nucleic acid amplification techniques.
  • a control mRNA showing little change in the 375' qPCR ratio with time relative to a sentienl mRNA would be MT-COl (RNA sequencing data and 375' Ratio provided in FIGURE 9).
  • amplification-based assays are used to measure the intactness and/or quantity of sentinel RNAs in a biological sample. As noted above, such assays can be used for control or standard determination and/or for biological sample evaluation. Such assays can rapidly assess RNA integrity in biospecimens prior to downstream gene expression analysis or diagnostic/prognostic testing. In some
  • the respective intactness and quantity of the one or more sentinel RNAs in the biological sample can be determined. Additionally or alternatively, in some embodiments, the intactness of sentinel RNA is determined independent of changes in RNA abundance.
  • RNA from a biological sample is isolated as described above. Additionally or alternatively in some embodiments, RNA from a biological sample is isolated using the 5 '-capped RNA isolation procedures described herein and/or total RNA is used. Additionally or alternatively, in some embodiments, a quantitative assessment of sentinel RNA present in a sample is determined. Additionally or alternatively, in some embodiments, in concert with this assessment, the 3 '-end degradation provides data required for determining whether the one or more sentinel RNAs are intact.
  • the amount of intactness e.g., non-degraded sentinel RNA
  • the amount of sentinel RNA degradation is about from 1, 5, 10, 20, 30, 40, or 50% to about 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more.
  • amplification-based procedures can also be performed to assess the ratio of 375' and/or 573' sentinel RNA in a biological sample.
  • ratios are measured at a single time point or at multiple, different time points.
  • Embodiments of the present technology include, for example, measuring 375' and/or 573' sentinel RNA degradation ratios in a biological sample at a single time.
  • sentinel RNA of a biological sample is measured at various time points, wherein the time points correlate to treatment of the sample, incubation at a specific temperature, etc., as described above.
  • sentinel RNAs in a biological sample possess decreasing 375' ratios as time increases. Additionally or alternatively, in some embodiments, sentinel RNAs processively decay with 3 '-5 polarity. Additionally or alternatively, in some embodiments, sentinel RNAs processively decay with 5 ' - 3 ' polarity. Additionally or alternatively , in some embodiments, sentinel RNAs processively decay in some embodiments with 3 '-5' polarity at a rapid rate.
  • a rapid decay rate is considered a > 20% decrease in sequencing reads on the 3' end (200 terminal nucleotides of the 3' untranslated end or UTR) compared to the 5' end of sentinel RNAs after 5 and 10 minutes at degradation (room temperature in the examples provided). Additionally or alternatively, in some
  • rapid decay rate is defined as a slope of 0.2 between 0 and 10 minutes of degradation.
  • the foregoing embodiments can be measured, for example, using one or more amplification procedures known in the art.
  • RNA and/or DNA can be amplified using nucleic acid amplification techniques well known in the art.
  • nucleic acid amplification techniques include, but are nor limited to, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative reverse transcriptase polymerase chain reaction PCR (qRT-PCR), and/or ligase chain reaction.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase polymerase chain reaction
  • qRT-PCR quantitative reverse transcriptase polymerase chain reaction PCR
  • ligase chain reaction See Abravaya, K., et al., Nucleic Acids Research, 23:675-682, (1995), branched DNA signal amplification, Urdea, M.
  • RNA reporters S., et al., AIDS, 7 (suppl 2):S11-S 14, (1993), amplifiable RNA reporters, Q-beta replication, transcription-based amplification, boomerang DNA amplification, strand displacement activation, cycling probe technology, isothermal nucleic acid sequence based amplification (NASBA).
  • NASBA isothermal nucleic acid sequence based amplification
  • PCR is used to amplify a sequence of interest, i.e., a sentinel RNA sequence.
  • PCR is a technique for making many copies of a specific template DNA and/or cDNA sequence.
  • the reaction can include multiple amplification cycles and is initiated using primer sequences that hybridize to the 5 ' and 3 ' ends of the sequence to be copied.
  • the amplification cycle includes an initial denaturation, and typically up to 50 cycles of annealing, strand elongation and strand separation (denaturation).
  • the DNA and/or cDNA sequence between the primers is copied.
  • primers can bind to the copied DNA and/or cDNA as well as the original template sequence, so the total number of copies increases exponentially with time.
  • PCR is performed as according to Whelan et al., J of Clin Micro, 33(3):556- 561 (1995). Briefly, a PCR reaction mixture includes two specific primers, dNTPs, approximately 0.25 U of Taq polymerase, and lx PCR Buffer.
  • Some methods of the present disclosure employ reverse transcription of RNA to cDNA.
  • the method of reverse transcription and amplification may be performed by previously published or recommended procedures.
  • Various reverse transcriptases may be used, including, but not limited to, MMLV RT, RNase H mutants of MMLV RT such as Superscript and Superscript II (Life Technologies, GIBCO BRL, Gaithersburg, Md.), AMV RT, and thermostable reverse transcriptase from Thermus thermophilus .
  • MMLV RT RNase H mutants of MMLV RT
  • AMV RT thermostable reverse transcriptase from Thermus thermophilus
  • one method which may be used to convert RNA to cDNA is the protocol adapted from the Superscript II Preamplification system (Life Technologies, GIBCO BRL, Gaithersburg, Md.; catalog no.
  • qPCR quantitative reverse transcription PCR
  • the methods may include amplifying multiple nucleic acids in sample, also known as “multiplex detection” or “multiplexing.”
  • multiplex PCR refers to PCR, which involves adding more than one set of PCR primers to the reaction in order to detect and quantify multiple nucleic acids, including nucleic acids from one or more target gene markers.
  • multiplexing with an internal control e.g., 18s rRNA, GADPH, or ⁇ -actin
  • an internal control e.g., 18s rRNA, GADPH, or ⁇ -actin
  • Primers can be developed for the amplification and/or quantification assays described herein, for example, using methods well known in the art, e.g., Roche's Universal Probe Library Assay Design Center (Basel, Switzerland). The skilled artisan is capable of designing and preparing primers that are appropriate for amplifying a target or marker sequence.
  • the length of the amplification primers depends on several factors including the nucleotide sequence identity and the temperature at which these nucleic acids are hybridized or used during in vitro nucleic acid amplification. The considerations necessary to determine a preferred length for an amplification primer of a particular sequence identity are well-known to a person of ordinary skill. For example, the length of a short nucleic acid or oligonucleotide can relate to its hybridization specificity or selectivity.
  • primers for detecting one or more sentinel RNA primers may be designed based on the cDNA sequence available for one or more sentinel RNAs including, but are not limited to, e.g., one or more of the following genes or gene products: EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orO, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP
  • the one or more sentinel RNA primers are provided.
  • primers are provided in the table below.
  • the primers are for one or both of GNB2L1 and TMBIM6.
  • the amplification reactions on the present technology may include a labeled primer or probe, thereby allowing detection of the amplification products corresponding to that primer or probe.
  • the amplification may include a multiplicity of labeled primers or probes; such primers may be distinguishably labeled, allowing the simultaneous detection of multiple amplification products.
  • a primer or probe is labeled with a fluorogenic reporter dye that emits a detectable signal. While a suitable reporter dye is a fluorescent dye, any reporter dye that can be attached to a detection reagent such as an oligonucleotide probe or primer is suitable for use in the invention.
  • Such dyes include, but are not limited to, Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Edans, Eosin, Erythrosin, Fluorescein, 6-Fam, Tet, Joe, Hex, Oregon Green, Rhodamine, Rhodol Green, Tamra, Rox, and Texas Red.
  • amplification is monitored using "real-time" methods.
  • Real time PCR allows for the detection and quantitation of a nucleic acid target.
  • a fluorescent dye which may be a double-strand specific dye, such as SYBR Green® I.
  • other fluorescent dyes e.g., FAM or HEX, may be conjugated to an oligonucleotide probe or a primer.
  • Various instruments capable of performing real time PCR are known in the art and include, for example, ABI Prism® 7900 (Applied Biosystems) and LightCycler® systems (Roche).
  • the fluorescent signal generated at each cycle of PCR is proportional to the amount of PCR product.
  • a plot of fluorescence versus cycle number is used to describe the kinetics of amplification and a fluorescence threshold level is used to define a fractional cycle number related to initial template concentration.
  • enzymatic replication and amplification methods include, but are not limited to, isothermal methods, rolling circle methods, Hot-start PCR, real-time PCR, Allele-specific PCR, Assembly PCR or Polymerase Cycling Assembly (PCA), Asymmetric PCR, Colony PCR, Emulsion PCR, Fast PCR, Real-Time PCR, nucleic acid ligation, Gap Ligation Chain Reaction (Gap LCR), Ligation-mediated PCR,
  • MLPA Multiplex Ligation-dependent Probe Amplification,
  • GEXL-PCR Gap Extension Ligation PCR
  • Q-PCR quantitative PCR
  • QRT-PCR Quantitative real-time PCR
  • multiplex PCR Helicase-dependent amplification
  • Intersequence-specific (ISSR) PCR Inverse PCR
  • LATE-PCR Linear-After-The-Exponential-PCR
  • MSP Methylation-specific PCR
  • Nested PCR Overlap-extension PCR
  • PAN- AC assay Reverse Transcription PCR (RT-PCR), Rapid Amplification of cDNA Ends (RACE PCR), Single molecule
  • SMA PCR amplification PCR
  • TAIL-PCR Thermal asymmetric interlaced PCR
  • Touchdown PCR long PCR
  • nucleic acid sequencing including DNA sequencing and RNA sequencing
  • transcription reverse transcription, duplication, DNA or RNA ligation, and other nucleic acid extension reactions known in the art.
  • 375 '-end expression ratios are generated for each test RNA transcript using formulas for quantification in real-time RT- PCR as described.
  • Pfaffl, M.W. "A new mathematical model for relative quantification in real-time qRT-PCR.” Nucleic acids research, 29, e45 (2001).
  • the formula is as follows:
  • the 375' ratio data equals the efficiency of the target qRT- PCR raised to the power of cycle threshold for the target control subtracted from the cycle threshold of the target sample. Additionally or alternatively, in some embodiments, the ascertained value is subsequently divided by the efficiency of the reference raised to the cycle threshold of the reference control subtracted from the cycle threshold of the reference sample. Additionally or alternatively, in some embodiments, an efficiency of two is employed for all calculations. In some embodiments, exact PCR efficiencies (generally between 1.8 and 2.2) may be calculated for both 5 ' and 3 ' end primer pairs to most accurately calculate 375' ratios.
  • the above formula target and reference are defined as the 3'- and 5 '-end amplicons of the same sentinel RNA, respectively.
  • Cycle thresholds and the 0 minute time point can serve as the cycle threshold control in some embodiments.
  • the sentinel RNAs described herein, as listed above can be grouped in non- limiting assay panels for use in the 375' qRT- PCR assays methods described herein.
  • at least from about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or 500 sentinel RNAs to about 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 1 ,000, or 10,000 sentinel RNAs are employed for the 3 5' qRT-PCR assays in accordance with the methods provided herein.
  • the 375' qRT-PCR assays described herein are analyzed in comparison to reference standards and/or control samples, from which standard curves were generated for specific tissues and/or assays, as described above. Additionally or alternatively, in some embodiments, the minimum number of sentinel RNAs selected for a 375' qRT-PCR assay to detect degradation of about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, or 90% of the niRNAs in a biological sample are be determined.
  • the minimum number of sentinel RNAs selected is about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, or 90% of the mRNAs in a biological sample. Additionally or alternatively, in some embodiments, the minimum number of sentinel RNAs selected is about 5% of the mRNAs in a biological sample.
  • >3 sentinel RNAs are used to determine the RNA integrity of a biospecimens sample.
  • the exact number of sentinels selected will be determined, in part, by the tissue being sampled as some tissues may have lower overall expression of some sentinels.
  • the number of sentinel RNAs selected is about 10.
  • a panel of sentinel RNAs includes one or more of the following: SERPINA3, B2M, ENOl , GNB2L1 , TMBIM6, PFN1 , MYL6, SAA2, JUNB and TIMP1. 8. Statistical Methods
  • a sentinel RNA may be present at an elevated amount or at a decreased amount in samples of patients having or suspected of having a disease or medical condition compared to a reference level.
  • a "difference of a level” may be a statistically significant difference. For example, a difference may be statistically significant if the measured level of the biomarker falls outside of about 1.0 standard deviations, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 stand deviations of the mean of any control or reference group.
  • statistics can be used to determine the validity of the difference or similarity observed between a patient's gene expression level and the reference level.
  • Exemplary statistical analysis methods are described in L.D. Fisher & G. vanBelle, Biostatistics: A Methodology for the Health Sciences (Wiley-lnterscience, NY, 1993). For instance, confidence ("/?") values can be calculated using an unpaired 2-tailed t test, with a difference between groups deemed significant if the p value is less than or equal to 0.05.
  • RNA protection assay RNA protection assay
  • RDA representation difference analysis
  • SAGE serial analysis of gene expression
  • LMF multiplex ligation-mediated amplification with the Luminex FlexMAP
  • WO 91/06678 Kwiatkowski, M., United States Patent Nos. US 6,255,475, US 6,309,836, and US 6639088 and EP1218391; Anazawa, T., et al., United States Patent No. 6242193; Ju, et al., United States Patent No. US 6,664,079; Tsien, R.Y., et al, International Patent Appl. No. WO 91/06678; and Dower, et al, International Patent Appl. No. WO 92/10587.
  • kits employed in the disclosed methods can be packaged into diagnostic kits and the like.
  • Diagnostic kits can include, for example, at least one or more primers specific for one or more sentinel RNAs. Additionally or alternatively, in some embodiments the kit includes nucleotide bases capable of being incorporated into an elongating oligonucleotide by a polymerase. Additionally or alternatively, in some embodiments, the bases are labeled. Additionally or alternatively, in some embodiments, specific labeling reagents can also be included in the kits as disclosed herein.
  • the kit can also contain other suitably packaged reagents and materials needed for amplification, for example, buffers, dNTPs, or polymerizing enzymes, and for detection analysis, for example, enzymes and solid phase extractants.
  • the kits comprise multiple amplification primer sets, wherein at least one of the primers is composed of a sequence complementary to at least a portion of one or more sentinel RNAs, such as, e.g., GNB2L1 and TMBIM6, or any other sentinel RNA as described herein.
  • the kits include controls and/or standards.
  • kits include one or more of the following (consistent with methods, reagents, and compositions discussed above):
  • components for sample purification including a lysis buffer with a chaotropic agent; a glass-fiber filter or column; an elution buffer; a wash buffer; an alcohol solution; and/or a nuclease inhibitor.
  • the components of the kits may be packaged either in aqueous media or in lyophilized form, for example, and will be provided in a suitable container.
  • the components of the kits provided herein may be provided as dried powder(s). Additionally or alternatively, in some embodiments, when reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent.
  • the solvent may also be provided in another container.
  • the container will generally include at least one vial, test tube, flask, bottle, syringe, and/or other container means, into which the solvent is placed, optionally aliquoted.
  • the kits also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other solvent.
  • Reagents useful for the disclosed methods can be stored in solution or can be lyophilized. When lyophilized, some or all of the reagents can be readily stored in microtiter plate wells for easy use after reconstitution. It is contemplated that any method for lyophilizing reagents known in the art would be suitable for preparing dried down reagents useful for the disclosed methods.
  • kits include control samples or standards, and/or control values or standards.
  • compositions, methods and kits described herein will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present methods and kits.
  • sentinel RNAs represent biomarkers that reflect of the quality of most mRNA contained in a biological sample.
  • Next generation RNA-sequencing was employed as described in the Examples below to analyze 5 '-capped mRNA recovered from human liver biospecimens which were thawed, and subsequently incubated at RT for increasing time periods.
  • candidate sentinel mRNAs were identified using sequencing data by calculating the RPKM ratio in the 3'- and 5 '-terminal ends of transcripts at varying times.
  • RNA samples were obtained with IRB approval and flash frozen with liquid nitrogen and stored at -80°C. Tissue samples were allowed to thaw at room temperature for 0, 5, 10, and 15 min before isolating total RNA.
  • Total RNA was isolated using Trizol® (Invitrogen, Carlsbad, CA) following standard methods. See, e.g., Choi et ah, "Purifying mRNAs with a high-affinity eIF4E mutant identifies the short 3' poly(A) end phenotype.” Proceedings of the National Academy of Sciences of the United States of America, , 7033-7038 (2003). The quality of total RNA was measured using a bioanalyzer from Agilent Technologies (Santa Clara, CA).
  • RNA polymerase II transcripts possessing a 5' m7GpppN cap were purified using a recombinant high affinity variant of eIF4E
  • eIF4EKl 19A which binds to the 5'-cap with at least a tenfold higher affinity than the wild- type protein.
  • Spivak-Kroizman et al. "Mutations in the S4-H2 loop of eIF4E which increase the affinity for m7GTP.”
  • Friedland et al "A mutant of eukaryotic protein synthesis initiation factor eIF4E(Kl 19A) has an increased binding affinity for both m7G cap analogues and eIF4G peptides.” Biochemistry, 44, 4546-4550 (2005).
  • GST-tagged eIF4EKl 19A protein bound to glutathione-agarose beads was used to purify 5'-capped RNA as described. See, e.g., Choi et al. (2003).
  • RNA polymerase II transcripts were analyzed by RNA sequencing with an Illumina Genome Analyzer 2 (GA2) and using standard protocols for preparing and sequencing libraries representing RNA samples. 5'-capped RNAs with rRNA removed (Ribominus® for RNA-seq kit, Invitrogen, Carlsbad, CA) were fragmented, reverse transcribed to cDNA, and adapters added to create a cDNA library. cDNAs with adapters were amplified by PCR using a Cluster Station (Illumina, San Diego, CA) and sequenced with an Illumina GA2.
  • GA2 Illumina Genome Analyzer 2
  • the bioinformatic criteria used to identify candidate sentinel RNAs were as follows: all genes - 36,615 genes in the Ensembl human genome 19 March 2009 build; expressed genes - 12,166 genes with 10 or more sequencing reads in the 15 minute sample; abundantly expressed genes - 4,877 genes with 50 or more reads in the 15 minute sample; abundantly expressed genes with significant 5' and 3' transcription - 706 genes with 25 or more reads in the terminal 200 bp of their 5' and 3' ends at 0 minutes and 25 or more reads in their 5' end after 5, 10, and 15 minutes; RNAs with an overall decrease in 375' ratios - 565 RNAs with a slope for the best fit linear line of ⁇ -0.2; and RNAs with progressive 3' to 5' degradation - 304 RNAs showed a difference in 375' ratio of > 0.2 between 0, 5, and 10 minute time points. After ten minutes half of these transcripts (148 or 49%) had ⁇ 25 reads in their
  • the cDNA was produced using random hexamers from two different preparations of RNA: 5 '-cap dependent RNA isolation and total RNA treated with
  • Ribominus® PCR was performed in 96-well plates in a final reaction volume of 20 ⁇ .
  • RNA Integrity Number (RIN) values of 9.5, 8.9, 7.9, and 6.7, respectively (see Figure 1). These values fall within the recommended guidelines for intact high quality RNA that would be examined by gene arrays, RNA sequencing (RNA-seq), and in clinical diagnostic or prognostic gene array testing. See, e.g., Schroeder et al., "The RIN: an RNA integrity number for assigning integrity values to RNA
  • the vertical bar represents the total number of reads for a 36 nucleotide sequence of isolated 5'- capped RNA, which was aligned with the corresponding genomic region, as provided (Hg 19, 2009).
  • Rapidly degrading transcripts such as, e.g., JUNB ⁇ see Figure 4B
  • 33%, 60%) and 78% reductions in JUNB 3' reads were observed after 5, 10 and 15 min, respectively.
  • PFN1 decreased by 39%>, 66%>, and 86%> after 5, 10 and 15 min, respectively (Figure 4A).
  • HIST1H1E see Figure 4C
  • MT-BCOl see Figure 4D
  • Candidate sentinel mRNAs were identified as described in Example 1, i.e., by employing RNA sequencing data and calculating the ratio of sequence reads in the last 200 3 '-end bases to the first 200 5 '-end bases (3' end/5' end ratio), at various time points. A total of 304 of candidate sentinel RNAs were identified as shown below in Table 2.
  • RNAs that reflect the integrity of most mRNAs in a sample is useful in developing improved and more rapid measures of RNA integrity in biospecimens.
  • 304 candidate sentinel mRNA transcripts were identified that were rapidly degraded in a 3' to 5' processive order, by mining the Pol II RNA-seq dataset described with specific criteria. These RNAs, or variants thereof, can be used to determine the integrity of a biological sample.
  • a candidate sentinel RNA is defined as a protein coding transcript that has specific expression criteria and shows a difference in 375' sequence reads of >0.2 between 0.5 and 10 minutes (see Materials and Methods).
  • mitochondrial transcript MT-BC01
  • HIST1H1E is another mRNA that showed very slow degradation with time ( Figure 4C).
  • Such transcripts provide useful controls.
  • qRT-PCR is employed as follows to facilitate the determination of biological sample quality via sentinel RNA analysis.
  • a standard curve is prepared using a fresh tissue of interest and incubating at room temperature for 0, 10, 20, 40, 60 minutes. Each time point is divided into three sets thereby allowing for in parallel analysis through standard gene microarrays, such as, e.g., Affymetrix, to measure mRNAs of the most expressed genes, while performing pair-wise differential gene expression analyses.
  • standard gene microarrays such as, e.g., Affymetrix
  • the number of genes that are determined to be differentially expressed are a direct measure of a "false positives" due to, e.g., sample degradation at the thresholds used to select the differentially expressed genes.
  • differentially expressed genes at a false discovery rate ("FDR") of 1% and 2X difference.
  • FDR false discovery rate
  • the FDR of a sentinel RNA is ⁇ 0.1.
  • threshold stringency is increased to accommodate acceptable FDR.
  • qRT-PCR is performed on tissue specific sentinel RNAs at each time point. Initially, this is performed at each time point with an unknown RNA set. The 375' ratio is calculated for each gene, at each time point, after converting the 3 '-end terminus and 5 '-end terminus cycle number to relative abundance. As such, at each time point an array of 375' ratios is obtained. Next the test sample is measured against a standard curve by calculating pair-wise Euclidian distances. The time points most closely associated with the test sample serve to score the sample in minutes of degradation.
  • RNA integrity is empirically determined for each application, e.g., using a fresh sample of a known quality of RNA, while subjecting such a sample to degradation using RNase and the like, and subsequently assaying the sample. 5.
  • RNA integrity is empirically determined for each application, e.g., using a fresh sample of a known quality of RNA, while subjecting such a sample to degradation using RNase and the like, and subsequently assaying the sample. 5.
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.
  • a range includes each individual member.
  • a group having 1-3 nucleotides refers to groups having 1, 2, or 3 nucleotides.
  • a group having 1-5 nucleotides refers to groups having 1, 2, 3, 4, or 5 nucleotides, and so forth.

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Abstract

Disclosed are methods, kits, and components for indentifying and detecting nucleic acid molecules, such as in a biological sample. The compositions, methods and kits are useful to assess the integrity of a biological sample.

Description

METHODS FOR DETERMINING THE INTEGRITY OF A
BIOLOGICAL SAMPLE
TECHNICAL FIELD
[0001] The present compositions, methods and kits relate broadly to the identification of nucleic acids for determining the overall quality of a biological test sample. In particular, the compositions, methods and kits relate to assessing the quantity and processive degradation of nucleic acid molecules as a measure of test sample integrity.
BACKGROUND
[0002] Numerous technologies such as gene arrays, multiplexed PCR, etc. facilitate the determination of nucleic acid expression levels, which can serve as diagnostic or prognostic indicators of disease. Such tests are clinically employed to aid in the selection of appropriate treatment regimens for patients with various disease indications, such as, e.g., cancer. As such, accurate measures of gene expression can be critical to ensure that the proper therapy is chosen or diagnosis is established. Biological test samples, however, are routinely collected, stored and/or processed under suboptimal conditions for maintaining intact oligonucleotides, e.g., samples may be subjected to room temperature for extended periods of time subsequent to collection. Furthermore, RNases are ubiquitous in biological samples and therefore samples containing RNA are susceptible to degradation. It follows that, if the quality of isolated RNA is compromised, i.e., from a degraded sample, then inaccurate expression profiles may result. Consequently, medical practitioners may prescribe improper treatment protocols or diagnoses based on inaccurate data.
SUMMARY
[0003] There are provided herein compositions, methods and kits for detecting and quantifying nucleic acid markers, which serve as a measure of biological sample integrity. The methods described herein measure the quantity and intactness, i.e., level of degradation, of representative RNAs as a conduit for determining the overall quality of a biological test sample. As described herein, in some embodiments, such representative RNAs are abundantly expressed and processively degrade with 3 '-5' polarity.
[0004] In some aspects, methods for determining the integrity of a biological sample are provided. In some embodiments, the methods include identifying one or more sentinel RNAs in the biological sample, determining the amount of degradation in the one or more sentinel RNAs, comparing the amount of degradation to a reference standard, and correlating the degradation to the integrity of the biological sample. In some embodiments, the biological sample includes one or more of blood, plasma, serum, lymph, mucus, sputum, tears, urine, stool, saliva, tissue, hair, animal cells, and plant cells.
[0005] Additionally or alternatively, in some embodiments, the one or more sentinel RNAs are mRNA. For example, in some embodiments, the one or more sentinel RNAs include one or more of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orf3, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT, SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, ALB, CLNS1A, ARF1, NDUFV1, RPL10A, SDC4, LASP1, ECHS1, C19orf63, FGG, TNFRSF1A, EIF4EBP1, CDC42EP1, RPNl, HLA-B, TFR2, SERPINA3, TMEMl l l, TMSB4X, AC025165.27, AMBP, ACATl, SEC61A1, BRP44L, CTSB, MARS, PCK1, DHCR24, KNG1, TUBA IB, SLC27A5, UBA1, FLU, RHOA, HNF4A, CTSD, IGFBP4, P4HB, LRRC59, SERPINA10, GC, LRPAP1, RPS5, DNAJC3, C4BPB, VTN, IGFBP1, RPN2, BCL2L1, TUBB6, LY6E, FLOT2, NANS, HPN, SERPINF2, SLC9A3R1, ARMET, IDH2, PSMC4, LAPTM4A, GPI, FURIN, APOB, APOH, XBP1, SERPINA11, GUSB, SEMA4B, HPD, KRT18,
TMEM176A, RPS2, LRP1, TGM2, FKBP11, TRIB1, SOD2, AC087521.10, BRI3, GABARAP, CRP, YWHAE, GAPDH, SERPINC1, ITIH1, HMOX1, CHI3L1, C14orf68, HPX, TPT1, HSP90B1, SERPINF1, H19, HSD17B10, ARHGDIA, GDI1, ACTB, KRT8, CFL1, AF235103.4, HABP2, PLA2G2A, ATP1A1, PRDX2, RPS3, DAD1, METTL7B, AXUD1, PSMD13, HM13, C19orfl0, MORF4L2, FNDC4, SRPR, MSN, SERPINB1, RPS4X, SAT1, TAGLN2, PSMB7, LAMP1, SERPINA1, CPS1, HSPE1, PSME1,
TMEM205, TUBB2C, C8B, RPS13, MLF2, RPL4, PMPCA, UQCRC1, LTBR, PEBP1, APOL1, ATP6V0E, TYMP, RPS9, FTL, AQP3, SERPINA7, LRG1, GSTK1, ETFB, GRINA, FGA, C21orf33, TCF25, EIF3K, CSTB, C5, CHAC1, EIF3G, IFITM1, APOA4, VPS28, POLD4, F2, ITGA5, DDX17, HSD17B2, SAT2, FGB, PDIA4, ACADVL, IGFBP2, EIF5A, TMBIM6, OAF, ACY1, PHB2, HLA-E, RHOD, ACOl 1498.7, CIB1, RPL12, GRHPR, RARRES2, TTR, GPX3, CD74, RPL30, HRG, RASD1, C9, SSR2, SHC1, C3, AZGP1, RRBP1, CALR, NDUFA4, GNB2L1, C19orf43, MDH2, GSTOl, LMAN1, APCS, PSMD4, SDS, PPIB, AP001453.6, BLOC1S1, RPL35, GADD45B, RPLP1, TMED9, AARS, SDF2L1, EPN1, PFN1, DUFS6, NDUFB9, GLTPD2, PSMB4, GCHFR, SPCS3, ID1, RABAC1, RTK , COX8A, PSMC5, GLTSCR2, SF3B5, RPS20, NDUFB2, NEDD8, JUNB, MYL6, EEF2, ARL5B, TOMM7, FAM96B, SAA2, RPL11, TRAPPC1, EDF1, Cl lorflO, ATP5D, RPL35A, GDI2, SURF4, RPL31, G0S2, SEC61G, NOLA3, SAA4, VAMP 8, IFITM3, C8G, and RPL37A. In some embodiments, the one or more sentinel RNAs are one or both of GNB2L1 and TMBIM6.
[0006] Additionally or alternatively, in some embodiments of the methods, the amount of degradation includes one or more selected from the group consisting of: 3 '-5' processive degradation, 5 '-3 ' processive degradation, a ratio of 3 '-5' processive degradation to 5 '-3' processive degradation, and a ratio of 5 '-3 ' processive degradation to 3 '-5' processive degradation. For example, in some embodiments, the 3 '-5 ' processive degradation is determined from 200 nucleotides from the 3 '-end of the one or more sentinel RNAs.
Additionally or alternatively, in some embodiments, the 5 '-3' processive degradation is determined from 200 nucleotides from the 5 '-end of the one or more sentinel RNAs.
[0007] Additionally or alternatively, in some embodiments, measuring is by RNA sequencing. For example in some embodiments, the measuring is by quantitative PCR. In some embodiments, for example, the quantitative PCR is performed using primers specific for the one or more sentinel RNAs including one or more of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orf3, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKNIA, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT, SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, ALB, CLNS1A, ARF1, NDUFV1, RPL10A, SDC4, LASP1, ECHS1, C19orf63, FGG, TNFRSF1A, EIF4EBP1, CDC42EP1, RPN1, HLA-B, TFR2, SERPINA3, TMEM111, TMSB4X, AC025165.27, AMBP, ACAT1, SEC61A1, BRP44L, CTSB, MARS, PCK1, DHCR24, KNG1, TUBA IB, SLC27A5, UBA1, FLU, RHOA, HNF4A, CTSD, IGFBP4, P4HB, LRRC59, SERPINA10, GC, LRPAP1, RPS5, DNAJC3, C4BPB, VTN, IGFBP1, RPN2, BCL2L1, TUBB6, LY6E, FLOT2, NANS, HPN, SERPINF2, SLC9A3R1, ARMET, IDH2, PSMC4, LAPTM4A, GPI, FURIN, APOB, APOH, XBP1, SERPINA11, GUSB, SEMA4B, HPD, KRT18, TMEM176A, RPS2, LRP1, TGM2, FKBP11, TRIB1, SOD2, AC087521.10, BRI3, GABARAP, CRP, YWHAE, GAPDH, SERPINC1, ITIH1, HMOX1, CHI3L1, C14orf68, HPX, TPT1, HSP90B1, SERPINF1, H19, HSD17B10, ARHGDIA, GDI1, ACTB, KRT8, CFL1, AF235103.4, HABP2, PLA2G2A, ATP1A1, PRDX2, RPS3, DAD1, METTL7B, AXUD1, PSMD13, HM13, C19orfl0, MORF4L2, FNDC4, SRPR, MSN, SERPINB1, RPS4X, SAT1, TAGLN2, PSMB7, LAMP1, SERPINA1, CPS1, HSPE1, PSME1, TMEM205, TUBB2C, C8B, RPS13, MLF2, RPL4, PMPCA, UQCRC1, LTBR, PEBP1, APOL1, ATP6V0E, TYMP, RPS9, FTL, AQP3, SERPINA7, LRG1, GSTK1, ETFB, GRINA, FGA, C21or03, TCF25, EIF3K, CSTB, C5, CHAC1, EIF3G, IFITM1, APOA4, VPS28, POLD4, F2, ITGA5, DDX17, HSD17B2, SAT2, FGB, PDIA4, ACADVL, IGFBP2, EIF5A, TMBIM6, OAF, ACYl, PHB2, HLA-E, RHOD, ACOl 1498.7, CIBl, RPL12, GRHPR, RARRES2, TTR, GPX3, CD74, RPL30, HRG, RASDl, C9, SSR2, SHC1, C3, AZGP1, RRBP1, CALR, NDUFA4, GNB2L1, C19orf43, MDH2, GSTOl, LMAN1, APCS, PSMD4, SDS, PPIB, AP001453.6, BLOC1S1, RPL35, GADD45B, RPLP1, TMED9, AARS, SDF2L1, EPN1, PFN1, DUFS6, NDUFB9, GLTPD2, PSMB4, GCHFR, SPCS3, ID1, RABAC1, RTKN, COX8A, PSMC5, GLTSCR2, SF3B5, RPS20, NDUFB2, NEDD8, JUNB, MYL6, EEF2, ARL5B, TOMM7, FAM96B, SAA2, RPL11, TRAPPCl, EDF1, Cl lorflO, ATP5D, RPL35A, GDI2, SURF4, RPL31, G0S2, SEC61G, NOLA3, SAA4, VAMP 8, IFITM3, C8G, and RPL37A. In some embodiments, the primers specific for the one or more sentinel RNAs are primers specific for one or both of GNB2L1 and TMBIM6.
[0008] Additionally or alternatively, in some embodiments, a reference standard is included. In some embodiments, the reference standard comprises a known amount of sentinel RNA degradation from a control biological sample. In some embodiments, the amount of degradation is measured at one or more time points.
[0009] Additionally or alternatively, in some embodiments, the indentifying comprises isolating 5 '-capped RNA in the biological sample and determining the level of degradation at one or more time points. [0010] In some embodiments, a sentinel R A panel is provided. In some embodiments, the panel includes SERPINA3, B2M, ENOl, GNB2L1, TMBIM6, PFN1, MYL6, SAA2, JUNB and TIMP1.
[0011] In some aspects, a method of identifying one or more sentinel RNAs in a biological sample is provided. In some embodiments, the method includes isolating RNA from one or more abundantly expressed genes in the biological sample, measuring processive degradation of the isolated RNA at one or more time points; and classifying the one or more abundantly expressed genes as the one or more sentinel RNAs based on the measuring. In some embodiments, the isolated RNA is 5 '-capped RNA. In some embodiments, the isolated RNA is mRNA.
[0012] Additionally or alternatively, in some embodiments, the processive degradation includes one or more of: 3 '-5' processive degradation, 5 '-3' processive degradation, a ratio of 3 '-5' processive degradation to 5 '-3' processive degradation, and a ratio of 5 '-3' processive degradation to 3 '-5 ' processive degradation. Additionally or alternatively, in some embodiments, the 3 '-5' processive degradation is from 200 nucleotides from the 3'- end of the one or more sentinel RNAs. Additionally or alternatively, in some embodiments, the 5 '-3' processive degradation is from 200 nucleotides from the 5 '-end of the one or more sentinel RNAs.
[0013] Additionally or alternatively, in some embodiments, a control or standard is included. In some embodiments, the control comprises RNA. In some embodiments, the processive degradation is rapid compared to the processive degradation from a control RNA.
[0014] In some embodiments, the measuring is by RNA sequencing. Additionally or alternatively, in some embodiments, the measuring is by quantitative PCR.
[0015] Additionally or alternatively, in some embodiments, the sentinel RNA includes one or more of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1,
AGT, EIF1, SCARB1, C20orf3, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1,
AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1,
ERGIC3, IBTK, YIFIA, ENOl, UGT2B4, GABARAPLl, KDELR2, RHBDD2, CD63, F9,
TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT,
SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, ALB, CLNS1A, ARF1, NDUFV1, RPL10A, SDC4, LASP1, ECHS1, C19orf63, FGG, TNFRSF1A, EIF4EBP1, CDC42EP1, RPNl, HLA-B, TFR2, SERPINA3, TMEMl l l, TMSB4X, AC025165.27, AMBP, ACATl, SEC61A1, BRP44L, CTSB, MARS, PCK1, DHCR24, KNG1, TUBA IB, SLC27A5, UBA1, FLU, RHOA, HNF4A, CTSD, IGFBP4, P4HB, LRRC59, SERPINA10, GC, LRPAP1, RPS5, DNAJC3, C4BPB, VTN, IGFBP1, RPN2, BCL2L1, TUBB6, LY6E, FLOT2, NANS, HPN, SERPINF2, SLC9A3R1, ARMET, IDH2, PSMC4, LAPTM4A, GPI, FURIN, APOB, APOH, XBP1, SERPINA11, GUSB, SEMA4B, HPD, KRT18,
TMEM176A, RPS2, LRP1, TGM2, FKBP11, TRIB1, SOD2, AC087521.10, BRI3, GABARAP, CRP, YWHAE, GAPDH, SERPINC1, ITIH1, HMOX1, CHI3L1, C14orf68, HPX, TPT1, HSP90B1, SERPINF1, H19, HSD17B10, ARHGDIA, GDI1, ACTB, KRT8, CFL1, AF235103.4, HABP2, PLA2G2A, ATP1A1, PRDX2, RPS3, DAD1, METTL7B, AXUD1, PSMD13, HM13, C19orfl0, MORF4L2, FNDC4, SRPR, MSN, SERPINB1, RPS4X, SAT1, TAGLN2, PSMB7, LAMP1, SERPINA1, CPS1, HSPE1, PSME1,
TMEM205, TUBB2C, C8B, RPS13, MLF2, RPL4, PMPCA, UQCRC1, LTBR, PEBP1, APOL1, ATP6V0E, TYMP, RPS9, FTL, AQP3, SERPINA7, LRG1, GSTK1, ETFB, GRINA, FGA, C21orf33, TCF25, EIF3K, CSTB, C5, CHAC1, EIF3G, IFITM1, APOA4, VPS28, POLD4, F2, ITGA5, DDX17, HSD17B2, SAT2, FGB, PDIA4, ACADVL, IGFBP2, EIF5A, TMBIM6, OAF, ACY1, PHB2, HLA-E, RHOD, ACOl 1498.7, CIB1, RPL12, GRHPR, RARRES2, TTR, GPX3, CD74, RPL30, HRG, RASD1, C9, SSR2, SHC1, C3, AZGP1, RRBP1, CALR, NDUFA4, GNB2L1, C19orf43, MDH2, GSTOl, LMAN1, APCS, PSMD4, SDS, PPIB, AP001453.6, BLOC1S1, RPL35, GADD45B, RPLP1, TMED9, AARS, SDF2L1, EPN1, PFN1, DUFS6, NDUFB9, GLTPD2, PSMB4, GCHFR, SPCS3, ID1, RABAC1, RTKN, COX8A, PSMC5, GLTSCR2, SF3B5, RPS20, NDUFB2, NEDD8, JUNB, MYL6, EEF2, ARL5B, TOMM7, FAM96B, SAA2, RPL11, TRAPPC1, EDF1, Cl lorflO, ATP5D, RPL35A, GDI2, SURF4, RPL31, G0S2, SEC61G, NOLA3, SAA4, VAMP 8, IFITM3, C8G, and RPL37A.
[0016] In some embodiments, the quantitative PCR is performed using primers specific for one or more sentinel RNAs. In some embodiments, the sentinel RNAs include one or more of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orf3, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIFIA, ENOl, UGT2B4, GABARAPLl, KDELR2, RHBDD2, CD63, F9, TRAMl, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT, SERPINA5, ATP5C1, RPLl 3 A, LBP, GSDMD, ALB, CLNS1A, ARF1, NDUFV1, RPL10A, SDC4, LASP1, ECHS1, C19orf63, FGG, TNFRSF1A, EIF4EBP1, CDC42EP1, RPN1, HLA-B, TFR2, SERPINA3, TMEM111, TMSB4X, AC025165.27, AMBP, ACAT1, SEC61A1, BRP44L, CTSB, MARS, PCK1, DHCR24, KNG1, TUBA IB, SLC27A5, UBA1, FLU, RHOA, HNF4A, CTSD, IGFBP4, P4HB, LRRC59, SERPINA10, GC, LRPAP1, RPS5, DNAJC3, C4BPB, VTN, IGFBPl, RPN2, BCL2L1, TUBB6, LY6E, FLOT2, NANS, HPN, SERPINF2, SLC9A3R1, ARMET, IDH2, PSMC4, LAPTM4A, GPI, FURIN, APOB, APOH, XBPl, SERPINAl l, GUSB, SEMA4B, HPD, KRT18, TMEM176A, RPS2, LRPl, TGM2, FKBP11, TRIB1, SOD2, AC087521.10, BRI3, GABARAP, CRP, YWHAE, GAPDH, SERPINC1, ITIH1, HMOX1, CHI3L1, C14orf68, HPX, TPT1, HSP90B1, SERPINF1, H19, HSD17B10, ARHGDIA, GDI1, ACTB, KRT8, CFL1, AF235103.4, HABP2, PLA2G2A, ATP1A1, PRDX2, RPS3, DAD1, METTL7B, AXUD1, PSMD13, HM13, C19orfl0, MORF4L2, FNDC4, SRPR, MSN, SERPINB1, RPS4X, SAT1,
TAGLN2, PSMB7, LAMP1, SERPINA1, CPS1, HSPE1, PSME1, TMEM205, TUBB2C, C8B, RPS13, MLF2, RPL4, PMPCA, UQCRC1, LTBR, PEBP1, APOL1, ATP6V0E, TYMP, RPS9, FTL, AQP3, SERPINA7, LRG1, GSTK1, ETFB, GRINA, FGA, C21orf33, TCF25, EIF3K, CSTB, C5, CHAC1, EIF3G, IFITM1, APOA4, VPS28, POLD4, F2, ITGA5, DDX17, HSD17B2, SAT2, FGB, PDIA4, ACADVL, IGFBP2, EIF5A, TMBIM6, OAF, ACY1, PHB2, HLA-E, RHOD, ACOl 1498.7, CIB1, RPL12, GRHPR, RARRES2, TTR, GPX3, CD74, RPL30, HRG, RASD1, C9, SSR2, SHC1, C3, AZGP1, RRBP1, CALR, NDUFA4, GNB2L1, C19orf43, MDH2, GSTOl, LMAN1, APCS, PSMD4, SDS, PPIB, AP001453.6, BLOC1S1, RPL35, GADD45B, RPLP1, TMED9, AARS, SDF2L1, EPN1, PFN1, DUFS6, NDUFB9, GLTPD2, PSMB4, GCHFR, SPCS3, ID1, RABAC1, RTKN, COX8A, PSMC5, GLTSCR2, SF3B5, RPS20, NDUFB2, NEDD8, JUNB, MYL6, EEF2, ARL5B, TOMM7, FAM96B, SAA2, RPLl 1, TRAPPCl, EDFl, Cl lorfTO, ATP5D, RPL35A, GDI2, SURF4, RPL31, G0S2, SEC61G, NOLA3, SAA4, VAMP 8, IFITM3, C8G, and RPL37A. Additionally or alternatively, in some embodiments, the primers specific for the one or more sentinel RNAs are primers specific for one or both of GNB2L1 and TMBIM6.
[0017] Additionally or alternatively, in some embodiments, the biological sample includes one or more of blood, plasma, serum, lymph, mucus, sputum, tears, urine, stool, saliva, tissue, hair, animal cells, and plant cells. [0018] In some aspects, kits for testing the integrity of a biological sample are provided. In some embodiments, the kit includes one or more primers specific for one or more sentinel RNAs, optionally, reagents for qPCR or RNA sequencing; and degradation reference standards.
[0019] In some embodiments, the kit the primers are specific for the one or more sentinel RNAs including one or more of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orO, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT, SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, ALB, CLNS1A, ARF1, NDUFV1, RPL10A, SDC4, LASP1, ECHS1, C19orf63, FGG, TNFRSF1A, EIF4EBP1, CDC42EP1, RPN1, HLA-B, TFR2, SERPINA3, TMEM111, TMSB4X, AC025165.27, AMBP, ACATl, SEC61A1, BRP44L, CTSB, MARS, PCKl, DHCR24, KNGl, TUBAIB, SLC27A5, UBAl, FLU, RHOA, HNF4A, CTSD, IGFBP4, P4HB, LRRC59, SERPINAIO, GC, LRPAP1, RPS5, DNAJC3, C4BPB, VTN, IGFBP1, RPN2, BCL2L1, TUBB6, LY6E, FLOT2, NANS, HPN, SERPINF2, SLC9A3R1, ARMET, IDH2, PSMC4, LAPTM4A, GPI, FURIN, APOB, APOH, XBP1, SERPINA11, GUSB, SEMA4B, HPD, KRT18,
TMEM176A, RPS2, LRP1, TGM2, FKBP11, TRIB1, SOD2, AC087521.10, BRI3, GABARAP, CRP, YWHAE, GAPDH, SERPINC1, ITIH1, HMOX1, CHI3L1, C14orf68, HPX, TPT1, HSP90B1, SERPINF1, H19, HSD17B10, ARHGDIA, GDI1, ACTB, KRT8, CFL1, AF235103.4, HABP2, PLA2G2A, ATP1A1, PRDX2, RPS3, DAD1, METTL7B, AXUD1, PSMD13, HM13, C19orfl0, MORF4L2, FNDC4, SRPR, MSN, SERPINB1, RPS4X, SAT1, TAGLN2, PSMB7, LAMP1, SERPINA1, CPS1, HSPE1, PSME1,
TMEM205, TUBB2C, C8B, RPS13, MLF2, RPL4, PMPCA, UQCRC1, LTBR, PEBP1, APOL1, ATP6V0E, TYMP, RPS9, FTL, AQP3, SERPINA7, LRG1, GSTK1, ETFB, GRINA, FGA, C21orf33, TCF25, EIF3K, CSTB, C5, CHAC1, EIF3G, IFITM1, APOA4, VPS28, POLD4, F2, ITGA5, DDX17, HSD17B2, SAT2, FGB, PDIA4, ACADVL, IGFBP2, EIF5A, TMBIM6, OAF, ACY1, PHB2, HLA-E, RHOD, ACOl 1498.7, CIB1, RPL12, GRHPR, RARRES2, TTR, GPX3, CD74, RPL30, HRG, RASD1, C9, SSR2, SHC1, C3, AZGP1, RRBP1, CALR, NDUFA4, GNB2L1, C19orf43, MDH2, GSTOl, LMAN1, APCS, PSMD4, SDS, PPIB, AP001453.6, BLOC1S1, RPL35, GADD45B, RPLP1, TMED9, AARS, SDF2L1, EPN1, PFN1, DUFS6, NDUFB9, GLTPD2, PSMB4, GCHFR, SPCS3, ID1, RABAC1, RTK , COX8A, PSMC5, GLTSCR2, SF3B5, RPS20, NDUFB2, NEDD8, JUNB, MYL6, EEF2, ARL5B, TOMM7, FAM96B, SAA2, RPL11, TRAPPC1, EDF1, Cl lorflO, ATP5D, RPL35A, GDI2, SURF4, RPL31, G0S2, SEC61G, NOLA3, SAA4, VAMP 8, IFITM3, C8G, and RPL37. Additionally or alternatively, in some embodiments, the kit includes primers specific for one or both of GNB2L1 and TMBIM6.
[0020] In some embodiments, the kit comprises PCR primers for sentinel RNAs
SERPINA3, B2M, ENOl, GNB2L1, TMBIM6, PFNl, MYL6, SAA2, JUNB and TIMP1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGURE 1 is a series of graphs showing RNA integrity analyses from frozen donor liver biospecimens that were incubated at room temperature (RT) at increase time intervals (0, 5, 10 or 15 minutes). Agilent electropherograms with the RNA integrity Number (RIN) are shown. RNA in 0, 5, and 10 minute time points were considered "good quality" having RIN values of > 7 and would be included in biospecimen gene expression studies based on current practices.
[0022] FIGURE 2 is a graph showing the percent of protein coding mRNAs with a >50% reduction in the total number of exonic sequencing reads or RPKM (reads per kilobase of gene per million reads) as samples (same as in FIGURE 1) were allowed to thaw at room temperature for 5, 10, and 15 minutes. A >50% reduction in sequencing reads was observed in 2.7% of mRNA transcripts by 5 minutes, 14% at 10 minutes, and 76% by 15 minutes.
[0023] FIGURE 3 is a graph showing the percent of protein coding mRNAs with >50, >75, and >90%> of their 0 minute sequencing reads after 5, 10, and 15 minutes.
[0024] FIGURE 4A-4D shows Integrated Genome Browser images showing RNA-seq reads of representative rapidly and slowly degrading mRNAs, isolated from flash frozen human liver directly (0 minutes), and after 5, 10, and 15 minutes at room temperature. Each vertical black bar represents the total number of reads for a 36 nucleotide sequence, originated from 5' capped RNA, and aligned with the corresponding genomic region.
Chromosomal coordinates of each gene are provided. Rapidly degrading transcripts, PFNl and JUNB (Panel A and B), showed a rapid decrease in number of reads, particularly at the 3' end, as time increased. JUNB showed a 33, 60, and 78% reduction in 3' reads after 5, 10 and 15 minutes, respectively. PFN1 showed a 39, 66, and 86% reduction after 5, 10 and 15 minutes). HIST1H1E (Panel C) and MT-BCOl (Panel D) maintained a higher number of 3' reads as time increased.
[0025] FIGURE 5 is a graph showing the integrity of protein coding Pol II RNAs as measured by sequencing 200 bp of their 3' and 5' ends. The RNA-seq data from the experiment described in Figure 1 and Materials and Methods was further analyzed with informatics programs. To calculate a 375' ratio for each RNA transcript the number of sequence reads from the last 200 bases (3' end) was divided by the reads from the first 200 bases (5' end) of each mRNA transcript and graphed according to time. Protein coding mRNAs with a minimum 5' end RPKM > 10 at all time points were included in the analysis to avoid confounding the analysis with genes with very low expression levels.
[0026] FIGURE 6 show pie charts of mappable Pol II RNA sequencing reads in intronic, exonic, and intergenic regions over time. The percentage of sequencing reads mapping to exonic, intronic, and intergenic regions at 0, 5, 10, and 15 minutes are shown schematically. With increasing time fewer reads were aligned to exonic regions, but not to intronic or intergenic RNAs. For each pie chart, the largest section is exons; the second largest section is introns and the smallest is intergenic.
[0027] FIGURE 7A-7J are graphs showing RNA-sequence and 375' qRT-PCR data of sentinel mRNAs with time. RNA-sequence data for five sentinel mRNAs mined from the database obtained from the biospecimens described in Figure 1 are presented. B2M (Panel A, B) SERPINA3 (Panel C, D), GNB2L1 (Panel E, F), TMBIM6 (Panel G, H) and ENOl (Panel I, J) showed a marked decrease in sequence reads, especially at the 3' end, as time increased. The RNA-sequence data is presented as in Figure 4. The 3 5' qRT-PCR analysis for each sentinel mRNA are shown in the panel below the sequencing data with increasing time. The 375' qRT-PCR data shown was obtained using 5' capped RNA and random hexamers to synthesize cDNA. Similar results were obtained from total RNA that had ribosomal RNA removed (Figures 10 and 11). Values represent mean 375' ratios ± SEM of three replicate qPCR assays at each time for three separate liver biospecimens (qPCR assay n=9). [0028] FIGURE 8A-8E are graphs showing RNA-sequence data of other sentinel mRNAs with time. 8A&B - TIMP1; 8C&D -PFN1; 8E&F -SAA2; 8G-H-MYL6;8I-J-JUNB . All graphs show gene orientation and sequencing reads 5 ' to 3 ' in IGB as in Figure 7 and below the sequencing reads the 375' qPCR ratio is given at each time (for one liver specimen; n=3 for qPCR assays at each time).
[0029] FIGURE 9A-9B are graphs showing examples of control mRNAs that exhibit little degradation with time. The RNA-sequence reads for MT-C01 (9 A) are shown as in Figure 7 and the 375' qPCR ratio (9B) is shown below. The RNA-sequence reads for HIST1H1E (Figure 4C) another sequence that shows slow or no degradation, is also shown herein.
[0030] FIGURE 10A-C are graphs showing 375 ' end ratios of B2M(A), GNB2L 1 (B) and TMB1M6(C) sentinel mRNAs at different time points (0, 5, 10 and 15 minutes) as determined by qPCR. Samples were total RNA that had ribosomal RNA removed. Values represent mean 375' ratios ± SEM of three replicates at each time.
DETAILED DESCRIPTION
[0031] Disclosed herein are methods and compositions for the identification,
amplification, and quantification of representative nucleic acids, e.g., sentinel RNAs, which can be used to quickly and accurately measure the usefulness and reliability of a biological sample by linking sentinel RNA integrity, e.g., intactness and/or degradation levels, to biological sample quality. The methods can be performed in a multiplex format which permits the determination of expression levels for two or more sentinel RNAs in a single reaction.
[0032] In practicing the present technology, many conventional techniques in molecular biology are used. These techniques are well-known and are explained in, e.g., Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et ah,
Molecular Cloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989); DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed. (1985); Oligonuchotide Synthesis, Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds. (1985); Transcription and Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the series, Meth. Enzymol, (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, NY, 1987); and et/z. Enzymol, Vols. 154 and 155, Wu & Grossman, and Wu, Eds., respectively.
[0033] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, unless otherwise stated, the singular forms "a," "an," and "the" include plural reference. Thus, for example, a reference to "an
oligonucleotide" includes a plurality of oligonucleotide molecules, and a reference to "a nucleic acid" is a reference to one or more nucleic acids. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. The definitions of certain terms as used in this specification are provided below.
[0034] As used herein, when referring to a numerical value, the term "about" means plus or minus 10% of the stated value unless otherwise indicated.
[0035] The terms "3'-end" or "3'-region" refer to the portion of a polynucleotide or oligonucleotide, e.g., RNA or DNA, located towards the 3'-end of the polynucleotide or oligonucleotide, and may or may not include the 3' most nucleotide(s) or moieties attached to the 3' most nucleotide of the same polynucleotide or oligonucleotide.
[0036] The terms "5'-end" or "5'-region" refer to the portion of a polynucleotide or oligonucleotide, e.g., RNA or DNA, located towards the 5' end of the polynucleotide or oligonucleotide, and may or may not include the 5' most nucleotide(s) or moieties attached to the 5' most nucleotide of the same polynucleotide or oligonucleotide.
[0037] As used herein, "amplification" or "amplifying" refers to the production of additional copies of a nucleic acid sequence. Amplification is typically performed by using, for example, polymerase chain reaction (PCR), reverse transcription RT-PCR, qPCR, qRT- PCR, etc., technologies and/or real time PCR and/or other technologies known in the art. The term "amplification reaction mixture" or "PCR mixture" refers to an aqueous solution comprising the various reagents used to amplify a target nucleic acid, e.g. , RNA, DNA, cDNA, etc., and the like. These may include enzymes, e.g., a thermostable polymerase, aqueous buffers, salts, amplification primers, target nucleic acid, and nucleotide triphosphates. Amplification may be exponential or linear. A nucleic acid to be amplified may be, for example, either RNA, DNA, cDNA, and the like or equivalents or complements thereof. The sequences amplified in this manner form an "amplicon." While the exemplary methods described hereinafter relate to amplification using PCR, qPCR, or qRT-PCR, numerous other methods are known in the art for amplification of nucleic acids, e.g., isothermal methods, rolling circle methods, etc. The skilled artisan will understand that these other methods may be used either in place of, or together with, PCR methods. See, e.g., Saiki, "Amplification of Genomic DNA" in PCR Protocols, Innis et ah, Eds.,
Academic Press, San Diego, CA 1990, pp. 13-20; Wharam et ah, Nucleic Acids Res., 2001, 29(11):E54-E54; Hafner et al, Biotechniques 2001, 30(4):852-6, 858, 860; Zhong et al, Biotechniques, 2001, 30(4):852-6, 858, 860.
[0038] The term "comparable" or "corresponding" in the context of comparing two or more samples, means that the same type of sample, e.g., a tissue sample, is used in the comparison. For example, nucleic acid degradation (e.g., DNA or RNA) in a biological sample can be compared to the same or different RNA or DNA of another biological sample. In some embodiments, comparable samples may be obtained from the same individual at different times. In other embodiments, comparable samples may be obtained from different individuals, e.g., a patient and a healthy individual. In general, comparable samples are normalized by a common factor. For example, body fluid samples are typically normalized by volume body fluid and cell-containing samples are normalized by protein content or cell count.
[0039] The terms "determining," "measuring," "assessing," and "assaying" are used interchangeably and include both quantitative and qualitative determinations. These terms refer to any form of measurement, and include determining if a characteristic, trait, or feature is present or not. Assessing may be relative or absolute. Assessing the presence of, for example, includes determining the amount of something present, as well as determining whether it is present or absent.
[0040] As used herein, the phrases "difference in the level of or "difference in the amount of refer to differences in the quantity of a particular biomarker, e.g., one or more nucleic acids, e.g., RNAs, in a sample as compared to a control or reference level. For example, the quantity of a particular RNA may be present at an elevated amount or at a decreased amount in samples of patients with a disease compared to a reference level. For example, in some embodiments, a "difference in the amount of may be a difference between the level of marker present in a sample as compared to a control. In some embodiments, "the difference in the amount of is at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%), at least about 25%, at least about 30%>, at least about 35%, at least about 40%>, at least about 50%), at least about 60%>, at least about 75%, at least about 80%> or more. In some embodiments, a "difference in the amount of can be a statistically significant difference between the level of the marker present in a sample as compared to a control. For example, a difference can be statistically significant if the measured level of the marker R A falls outside of about 1.0 standard deviations, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 stand deviations of the mean of any control or reference group.
[0041] As used herein, the terms "gene expression" or "expression" refer to the process of converting genetic information encoded in a gene into RNA, e.g., total RNA, mRNA, miRNA, rRNA, tRNA, or snRNA, through transcription of the gene, i.e., via the enzymatic action of an RNA polymerase, and for protein encoding genes, into protein through translation of mRNA. Gene expression can be regulated at many stages in the process. Up- regulation or activation refers to regulation that increases the production of gene expression products, i.e., RNA or protein, while down-regulation, repression or knock-down refers to regulation that decrease production. Molecules, e.g., transcription factors that are involved in up-regulation or down-regulation are often called activators and repressors, respectively.
[0042] As used herein, "nucleic acid," "nucleotide sequence," or "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof and to naturally occurring or synthetic molecules. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, or to any DNA-like or RNA-like material, including natural and/or non-natural bases.
[0043] As used herein, "microarray", "gene panel", "gene array", "array" and/or "tissue microarray" refers to an arrangement of a collection of nucleic acids, e.g., nucleotide sequences in a centralized location. Arrays can be on a solid substrate, such as a glass slide, or on a semi-solid substrate, such as nitrocellulose membrane. The nucleotide sequences can be DNA, RNA, or any combination or permutations thereof. The nucleotide sequences can also be partial sequences or fragments from a gene, primers, whole gene sequences, non-coding sequences, coding sequences, published sequences, known sequences, or novel sequences. Tissue microarrays are well known in the art and can be performed as described. See e.g., Camp, R. L., et al, J Clin Oncol, 26, 5630-5637 (2008).
[0044] As used herein, a "primer" for amplification is an oligonucleotide that specifically anneals to a target or marker nucleotide sequence and forms a substrate for a nucleic acid polymerase. The term primer includes "primer pairs" required to amplify a nucleic acid sequence. The 3' nucleotide of the primer can be complementary, identical, and/or hybridize to a target or marker sequence at a corresponding nucleotide position for optimal primer extension by a polymerase. As used herein, a "forward primer" is a primer that anneals to the anti-sense strand of double stranded DNA (dsDNA) or an appropriate cDNA or RNA or other nucleic acid with similar polarity. A "reverse primer" anneals to the sense- strand of dsDNA or an appropriate cDNA or RNA or other nucleic acids with similar polarity.
[0045] The term "prognosis" as used herein refers to a prediction of the probable course and outcome of a clinical condition or disease. A prognosis is usually made by evaluating factors or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease. The phrase "determining the prognosis" as used herein refers to the process by which the skilled artisan can predict the course or outcome of a condition in a patient. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy. Instead, the skilled artisan will understand that the term "prognosis" refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition. The terms "favorable prognosis" and "positive prognosis," or "unfavorable prognosis" and "negative prognosis" as used herein are relative terms for the prediction of the probable course and/or likely outcome of a condition or a disease. A favorable or positive prognosis predicts a better outcome for a condition than an unfavorable or negative prognosis. In a general sense, a "favorable prognosis" is an outcome that is relatively better than many other possible prognoses that could be associated with a particular condition, whereas an unfavorable prognosis predicts an outcome that is relatively worse than many other possible prognoses that could be associated with a particular condition. Typical examples of a favorable or positive prognosis include a better than average cure rate, a lower propensity for metastasis, a longer than expected life expectancy, differentiation of a benign process from a cancerous process, and the like. For example, a positive prognosis is one where a patient has a 50% probability of being cured of a particular disease, e.g., cancer, after treatment, while the average patient with the same cancer has only a 25% probability of being cured.
[0046] As used herein, the terms "reference level," "reference standard," "control" or "control sample" are used interchangeably and refer to a sample having a level or amount of a substance which may be of interest for comparative purposes. For example, in some embodiments, a reference level is the average of the amount of, and/or rate of, intact and/or degraded nucleic acid (e.g., sentinel RNA) from one or more biological samples taken from a control population of one or more healthy (disease-free) subjects. In some embodiments, the reference level is the amount of, and/or rate of, intact and/or degraded nucleic acid from the same subject at a different time, e.g., prior to the subject developing the disease and/or prior to and/or after and/or during therapy, and/or before and after a sample preparation procedure, and/or before and after sample storage. In general, samples are normalized by a common factor. For example, body fluid samples are normalized by volume body fluid and cell-containing samples are normalized by protein content or cell count.
[0047] As used herein, the term "sample," "biological sample," "test sample," "clinical sample," "laboratory sample," and/or "biospecimen" are used interchangeably and in the broadest sense. A sample may include a bodily tissue or a bodily fluid including but not limited to tissue samples, blood (or a fraction of blood such as plasma or serum), lymph, mucus, tears, urine, stool and saliva. A sample may include an extract from an animal or plant cell, a chromosome, organelle, or a virus. A sample may be a "cell-free" sample, meaning that the volume of cells in the sample are less than about 2% of the total sample volume (preferably less than about 1% of the total sample volume). In some embodiments, a sample may comprise RNA, e.g., mRNA or cDNA, any of which may be amplified to provide amplified nucleic acid. For example, a sample may include nucleic acid in solution or bound to a substrate, e.g. , as part of a microarray. A sample may be obtained from any subject or any patient.
[0048] As used herein, "sample integrity" or "sample quality" or "overall quality" are used interchangeably and refer the overall "health" of a sample as it relates to the amount, level, intactness, and/or rate of nucleic acid degradation, e.g., RNA degradation, therein. The "sample integrity" or "sample quality" or "overall quality" can also refer to the ability of the sample to accurately and/or reliably reflect the results for a given assay. Sample integrity or quality, however, is independent of the accuracy or reliability of any particular assay. For example, when employing a gene expression panel, a biological sample may have "optimal" or "good" quality or integrity if the sample possesses the nucleic acids which may be expressed and/or are expressed, e.g., one or more specific mR As. In contrast, when employing a gene expression panel, a biological sample may have
"suboptimal" or "poor" quality or integrity if the sample does not possess the nucleic acids which may be expressed and/or are expressed, but have since been degraded, or include degradation products of such nucleic acids.
[0049] As used herein, the term "subject" and "patient" are used interchangeably and refer to a mammal, such as a human, but can also be another animal such as a domestic animal, e.g., a dog, cat, or the like, a farm animal, e.g., a cow, a sheep, a pig, a horse, or the like, or a laboratory animal, e.g., a monkey, a rat, a mouse, a rabbit, a guinea pig, or the like.
[0050] As used herein, "sentinel RNA" or "sentinel RNAs" refer to one or more RNA molecules capable of serving as a biomarker for the quality of RNA, such as mRNA, in a biological sample. In some embodiments, the sentinel RNA serves as a biomarker for the quality of RNA, such as mRNA, in a biological sample at a given time point.
I. Overview
[0051] Clinical biospecimens are routinely analyzed to evaluate gene expression in diseased patients. Such gene expression analyses, however, are only effective as diagnostic or prognostic tools if they can accurately reflect the gene expression profile, e.g., the amount or level of expressed RNA, in the test sample. Surrogate markers, such as, for example, ribosomal RNA (rRNA) have been employed to indirectly assess RNA quality in a biological sample. See, e.g., Schroeder et al, "The RIN: an RNA integrity number for assigning integrity values to RNA measurements." BMC molecular biology, 7, 3 (2006). In this respect, the quality of test sample RNA is measured as a function of intact 18s and 28s rRNA to generate a RNA integrity number (RIN). Id. However, because RIN accuracy relies on an indirect measure for predicting RNA polymerase II (Pol II)/mRNA transcript integrity, there are inherent disadvantages to such a model.
[0052] Typically, a RIN of > 7 is considered to be "intact" RNA. See, e.g. , Auer et al. , "Chipping away at the chip bias: RNA degradation in microarray analysis." Nature genetics, 35, 292-293 (2003). RIN values of less than 7 may however produce spurious microarray results due to reduced transcript probe length and decreased yields prior to hybridization. See Thompson et al., "Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA." BMC biotechnology, 7, 57 (2007). Likewise, dispersion tree analyses show that some samples with RIN scores of less than 7.8 have been shown to differ in their gene expression profiles. See Copois et al, " Impact of RNA degradation on gene expression profiles: assessment of different methods to reliably determine RNA quality." Journal of
biotechnology, 111, 549-559 (2007). In fact, up to 30% of RNA transcripts possessing RIN scores of greater than 7.8 can be degraded prior to use in hybridization assays. Popova et al, "Effect of RNA quality on transcript intensity levels in microarray analysis of human post-mortem brain tissues." BMC genomics, 9, 91 (2008).
[0053] Other methods known in the art, e.g., RQS (RNA Quality Score) measurements and degradometer interrogation, for determining RNA integrity, suffer from similar deficiencies. See Thompson et al, "Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA." BMC biotechnology, 7, 57. (2007); Copois et al. (2007); and Popova et al. (2008). Many of these methods entail genome-based expression studies premised on the preparation of complemetary nucleic acids with intact 3 '-end RNA. RNA degradation models
nevertheless indicate that many RNAs are progressively degraded from the 3'-end, starting with deadenylation, as described herein. See also Wiederhold et ah, "Cytoplasmic deadenylation: regulation of mRNA fate." Biochem Soc Trans, 38, 1531-1536. (2010). Indeed, while performing RNA sequencing (RNA-seq) studies of 5'-methyl guanosine capped (5 '-capped or 5'-m7GpppN) RNA from a biological sample, the present inventors discovered that a surprisingly large percentage of mRNA transcripts were processively degraded from the 3 '-end after short room temperature (RT) incubations, e.g., 10 minutes (min).
[0054] Accordingly, by isolating 5'-m7GpppN capped RNA, the degree of degradation in a biological sample can be quantitatively determined using techniques known in the art, e.g., PCR, RT-PCR, and/or qRT-PCR, while avoiding the inconsistencies associated with 3 '-end RNA isolation. Thus, in order to develop superior measures of Pol II transcript integrity, and therefore biological sample quality, the present technology is directed to the
identification of 5'-m7GpppN capped, abundantly expressed and rapidly degrading RNAs, termed sentinel RNA, which can be used to accurately measure the quality or integrity of a biological sample. Candidate sentinel RNA degradation, and the concomitant level of intactness, are assessed to identify abundantly expressed genes, which degrade at a rapid rate, thereby allowing for the absolute and/or relative determination of 3'- and/or 5'-end decay of these candidate RNAs. Taken together, such determinations provide sentinel RNAs as a tool for assessing the overall quality of a biological sample, prior to, for example, clinical assessment and/or gene array analysis.
II. Sentinel RNAs
[0055] As used herein, "sentinel RNA" or "sentinel RNAs" refer to one or more RNA molecules capable of serving as a biomarker for the quality of RNA, such as mRNA, in a biological sample. In some embodiments, sentinel RNA serve as biomarkers for the quality of RNA, such as mRNA, in a biological sample at a given time point. As such, sentinel RNA imparts a mechanism for determining the integrity or quality of a biological sample. Typically, sentinel RNAs are abundantly expressed, e.g. , approximately two orders of magnitude above background expression, in a variety of tissues and cell types such as, but not limited to, e.g., one or more of muscle cells or tissue, epithelial cells or tissue, endothelia cells or tissue, organ cells or tissue, stem cells or tissue, umbilical vessel cells or tissue, corneal cells or tissue, cardiomyocytes, aortic cells or tissue, corneal epithelial cells or tissue, aortic endothelial cells or tissue, fibroblasts, hair cells or tissue, keratinocytes, melanocytes, adipose cells or tissue, bone cells or tissue, osteoblasts, airway cells or tissue, microvascular cells or tissue, mammary cells or tissue, vascular cells or tissue,
chondrocytes, placental cells or tissue, plant cells, and the like. In some embodiments, one or more sentinel RNAs are tissue specific. Additionally or alternatively, in some
embodiments, one or more sentinel RNAs do not possess a specific tropism.
[0056] sentinel RNAs are typically degraded at a rapid, or comparatively measurable, rate, e.g., not degradation resistant. Therefore, sentinel RNAs include, but are not limited to, biologically active or expressed RNA molecules, such as, e.g., exonic RNA (exons), messenger RNA (mRNA), microRNA (miRNA), transfer RNA (tRNA), and/or any RNA molecule possessing a 5'-m7 GpppN cap. In some embodiments, sentinel RNAs include, for example, biologically inert RNAs, such as, e.g., intronic RNA (introns), intergenic RNA, and the like, or any RNA species derived from the aforementioned classes of RNAs by metabolic processes.
[0057] In some embodiments, sentinel RNAs include, but are not limited to, e.g., RNAs from one or more of the following genes or gene products: EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orO, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT, SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, ALB, CLNS1A, ARF1, NDUFV1, RPL10A, SDC4, LASP1, ECHS1, C19orf63, FGG, TNFRSF1A, EIF4EBP1, CDC42EP1, RPN1, HLA-B, TFR2, SERPINA3, TMEM111, TMSB4X, AC025165.27, AMBP, ACAT1, SEC61A1, BRP44L, CTSB, MARS, PCK1, DHCR24, KNG1, TUBA IB, SLC27A5, UBA1, FLU, RHOA, HNF4A, CTSD, IGFBP4, P4HB, LRRC59, SERPINA10, GC, LRPAP1, RPS5, DNAJC3, C4BPB, VTN, IGFBP1, RPN2, BCL2L1, TUBB6, LY6E, FLOT2, NANS, HPN, SERPINF2, SLC9A3R1, ARMET, IDH2, PSMC4, LAPTM4A, GPI, FURIN, APOB, APOH, XBP1, SERPINA11, GUSB, SEMA4B, HPD, KRT18, TMEM176A, RPS2, LRP1, TGM2, FKBP11, TRIB1, SOD2, AC087521.10, BRI3, GABARAP, CRP, YWHAE, GAPDH, SERPINCl, ITIHl, HMOXl, CHI3L1, C14orf68, HPX, TPT1, HSP90B1, SERPINF1, H19, HSD17B10, ARHGDIA, GDIl, ACTB, KRT8, CFLl, AF235103.4, HABP2, PLA2G2A, ATPlAl, PRDX2, RPS3, DAD1, METTL7B, AXUD1, PSMD13, HM13, C19orfl0, MORF4L2, FNDC4, SRPR, MSN, SERPINB1, RPS4X, SAT1, TAGLN2, PSMB7, LAMP1, SERPINA1, CPS1, HSPE1, PSME1, TMEM205, TUBB2C, C8B, RPS13, MLF2, RPL4, PMPCA, UQCRC1, LTBR, PEBP1, APOL1, ATP6V0E, TYMP, RPS9, FTL, AQP3, SERPINA7, LRG1, GSTK1, ETFB, GRINA, FGA, C21or03, TCF25, EIF3K, CSTB, C5, CHAC1, EIF3G, IFITMl, APOA4, VPS28, POLD4, F2, ITGA5, DDX17, HSD17B2, SAT2, FGB, PDIA4, ACADVL, IGFBP2, EIF5A, TMBIM6, OAF, ACYl, PHB2, HLA-E, RHOD, ACOl 1498.7, CIBl, RPL12, GRHPR, RARRES2, TTR, GPX3, CD74, RPL30, HRG, RASDl, C9, SSR2, SHC1, C3, AZGP1, RRBP1, CALR, NDUFA4, GNB2L1, C19orf43, MDH2, GSTOl, LMAN1, APCS, PSMD4, SDS, PPIB, AP001453.6, BLOC1S1, RPL35, GADD45B, RPLP1, TMED9, AARS, SDF2L1, EPN1, PFN1, DUFS6, NDUFB9, GLTPD2, PSMB4, GCHFR, SPCS3, ID1, RABAC1, RTKN, COX8A, PSMC5, GLTSCR2, SF3B5, RPS20, NDUFB2, NEDD8, JUNB, MYL6, EEF2, ARL5B, TOMM7, FAM96B, SAA2, RPL11, TRAPPCl, EDF1, Cl lorflO, ATP5D, RPL35A, GDI2, SURF4, RPL31, G0S2, SEC61G, NOLA3, SAA4, VAMP 8, IFITM3, C8G, and/or RPL37A, and the like.
[0058] In some embodiments, the one or more sentinel RNAs are one or both of GNB2L1 and TMBIM6. The sentinel RNAs of the present technology can be directly or indirectly assayed. In some embodiments, one or more sentinel RNAs are used as a template for quantitative amplification, e.g., qRT-PCR, according to the methods disclosed herein.
Moreover, methods for identifying sentinel RNAs are provided herein.
[0059] Additionally or alternatively, the sentinel RNAs described herein, as listed above, can be grouped in non-limiting assay panels for use in the methods described herein. In some embodiments, at least from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or 500 sentinel RNAs to about 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 1,000, or 10,000 sentinel RNAs are group into arrays in accordance with the methods provided herein. Additionally or alternatively, in some embodiments, the one or more sentinel RNAs are assayed in a multiplexed format.
Biological Samples
A. Collection and Preparation [0060] The compositions, methods and kits described herein may be used to detect nucleic acids associated with various genes using a biological sample obtained from a subject. The biological sample may be from any organism that possesses endogenous nucleic acid (RNA and/or DNA). Biological samples can be obtained by standard procedures and can be used immediately or stored, under conditions appropriate for the type of biological sample, for later use. Methods of obtaining biological samples are well known to those of skill in the art and include, but are not limited to, aspirations, tissue sections, swabs, drawing of blood or other fluids, surgical or needle biopsies, and the like. Additionally or alternatively, in some embodiments, biological samples are obtained pursuant to the instructions or recommendations of a bioassay manufacturer (e.g., MammaPrint™, ColoPrint™, etc.) with which the sample is to be tested.
[0061] The starting material for the assays disclosed herein typically include, but are not limited to, one or more clinical samples, which are suspected to contain RNA, e.g., total cellular RNA, and/or one or more sentinel RNAs. The sample may be obtained from a subject or patient. The biological sample, in some embodiments, is a cell-containing liquid or a tissue. Samples may include, but are not limited to, biopsies, blood, blood cells, bone marrow, fine needle biopsy samples, peritoneal fluid, amniotic fluid, plasma, pleural fluid, saliva, semen, serum, tissue or tissue homogenates, frozen or paraffin sections of tissue. Samples may also be processed, such as sectioning of tissues, fractionation, purification, or cellular organelle separation. In some embodiments, the biological sample includes one or more of blood, plasma, serum, lymph, mucus, sputum, tears, urine, stool, saliva, tissue, hair, animal cells, and plant cells.
[0062] In some embodiments, the sample may contain cells, tissues or fluid obtained from a patient suspected being afflicted with a disease such as, but not limited to, e.g., exogenous diseases, including, but not limited to, bacterial, fungal, prion, and/or viral diseases, e.g., chronic viral hepatitis, and/or endogenous diseases, such as, but not limited to, cancers including, but not limited to, e.g., lymphatic cancers, hematologic cancers, breast cancer, liver cancer, lung cancer, prostate cancer, gastric cancer, endometrial cancer, salivary gland cancer, adrenal cancer, non-small cell lung cancer, pancreatic cancer, renal cancer, ovarian cancer, peritoneal cancer, head and neck cancer, bladder cancer, colorectal cancer, glioblastomas, hematologic tumors, multiple myeloma, acute myelogenous leukemia, and/or colon cancer, and/or metastatic cancers of the same. [0063] Methods for isolating a particular cell from other cells in a sample include, but are not limited to, Fluorescent Activated Cell Sorting (FACS) as described, for example, in Shapiro, Practical Flow Cytometry, 3rd edition Wiley-Liss; (1995), density gradient centrifugation, or manual separation using micromanipulation methods with microscope assistance. Exemplary cell separation devices that are useful in the invention include, without limitation, a Beckman JE-6 centrifugal elutriation system, Beckman Coulter EPICS ALTRA computer-controlled Flow Cytometer-cell sorter, Modular Flow Cytometer from Cytomation, Inc., Coulter counter and channelyzer system, density gradient apparatus, cytocentrifuge, Beckman J-6 centrifuge, EPICS V dual laser cell sorter, or EPICS PROFILE flow cytometer. A tissue or population of cells can also be removed by surgical techniques.
[0064] A biological sample can be prepared for use in the methods of the present invention by lysing a cell that contains one or more desired nucleic acids, e.g., total RNA, mRNA, 5 '-capped RNA, and/or sentinel RNAs. For example, in some embodiments, a cell is lysed under conditions that substantially preserve the integrity of the desired nucleic acid. For example, cells can be lysed or subtractions obtained under conditions which stabilize RNA and/or DNA. Such conditions include, for example, cell lysis in strong denaturants, including chaotropic salts such as guanidine thiocyanate, ionic detergents such as sodium dodecyl sulfate, organic solvents such as phenol, high lithium chloride concentrations or other conditions known in the art to be effective in limiting the activity of endogenous RNases during RNA purification. Additionally, relatively undamaged nucleic acids such as RNA can be obtained from a cell lysed by an enzyme that degrades the cell wall. Cells lacking a cell wall either naturally or due to enzymatic removal can also be lysed by exposure to osmotic stress. Other conditions that can be used to lyse a cell include exposure to detergents, mechanical disruption, sonication, heat, pressure differential such as in a French press device, or Dounce homogenization.
[0065] In some embodiments, the nucleic acids are separated from proteins and sugars present in the original sample and/or a sample that was subjected to cell lysis. Any purification methods known in the art may be used in the context of the present invention. Those skilled in the art will know or be able to readily determine methods for isolating nucleic acid from a cell, fluid or tissue, such as those described in Sambrook et ah,
Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001) or in Ausubel et ah, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998). Nucleic acid sequences in the sample can subsequently be amplified using in vitro amplification, such as PCR or qPCR. Typically, compounds that may inhibit polymerases are removed from the nucleic acids.
[0066] Additionally or alternatively, in some embodiments, the biological samples {e.g., cells, tissues, fluids, nucleic acid, etc. for testing) are prepared pursuant to the instructions or recommendations of a bioassay manufacturer {e.g., MammaPrint™, ColoPrint™, etc.) with which the sample is to be tested.
B. Methods for Isolating RNA
[0067] Additionally or alternatively, in some embodiments, RNA isolation and/or extraction (RNA purification) is required for a variety of biological assays. Gene expression arrays, RNA sequencing technologies, and tiling arrays, for example, are fundamental tools for developing diagnostic biomarkers of disease, which may require RNA purification. Time consuming annotation of expressed genes and RNA quality assurance in patient samples, however, underlie the problems associated with these approaches.
Moreover, some gene expression arrays, e.g., Affymetrix®, rely on degradation ratios of "housekeeping" genes to estimate complementary RNA (cRNA) synthesis efficiency and mRNA quality. For these assays, transcript signal values are calculated from probes annealed within 500 bases of the 5'- and/or 3'-end of genes such as, e.g., GAPDH and ACTB. The quality of mRNA, to this end, is measured as a function of the 375' ratio, wherein quotients of approximately three denote equal amounts of cDNA synthesis from the 5'- and 3 '-ends.
[0068] Such methods nevertheless typically isolate RNA transcripts from the 3 '-end, via oligo(dT) primers, which hybridize to the poly(A) terminus. In this regard, 5 '-end RNA levels can be underestimated due to 3 '-end degradation and/or the inability of a reverse transcriptase to processively reach the 5 '-end, i.e., when cDNA synthesis is required.
Consequently, the aforementioned assays may impart inaccurate RNA integrity
determinations, and, in turn, provide for inaccuracies with respect to the overall quality of a biological sample. These inaccuracies can be curtailed by properly isolating test sample RNA using the 5 '-capped purification techniques described herein.
[0069] Along these lines, the predominant forms of diagnostic and/or prognostic RNA are typically associated with RNA Pol II transcription products, which possess a 5'-m7GpppN cap (5 '-cap). Such RNAs include, but are not limited to, for example, exonic mRNA (exons), protein coding mRNA, micro-RNA (miRNA) precursors, and other RNAs which may not encode for specific proteins. See Cai et al, "Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs." RNA, 10, 1957- 1966 (2004). Accordingly, in some embodiments, the isolation and analysis of 5'-capped RNA enables the accurate identification and evaluation of sentinel RNAs, their intactness and/or the 3 -5' and 5 '-3' degradation that occurs in Pol II transcripts because such methods obviate the inconsistencies associated with 3' -RNA isolation.
[0070] Moreover, at least 10-20 times more RNA transcripts are present in eukaryotic cells than are currently annotated, yet many appear to lack 3'-poly(A) ends and a majority of these RNAs are Pol II transcripts. See Durtrow et al. (2008). These RNAs likely encode proteins and may also represent non-coding regulatory RNAs. In one aspect, the present technology therefore provides an efficient method of isolating Pol II transcripts by binding their 5 '-caps with a RNA cap binding protein and/or a high-affinity variant of the RNA cap binding protein (4EK119A). See, e.g., Choi et al, "Purifying mRNAs with a high-affinity eIF4E mutant identifies the short 3' poly(A) end phenotype." Proceedings of the National Academy of Sciences of the United States of America, , 7033-7038 (2003). Other methods for purifying RNA known in the art are not excluded with respect to the present methods and can additionally or alternatively be employed, as necessary.
[0071] The skilled artisan will readily appreciate that total RNA can be isolated from cells, tissues, biological samples, and the like, using RNA isolation methods known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York (2001) or in Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998). RNA sequences in the test sample can subsequently be amplified using in vitro amplification, such as, for example, qRT-PCR. Typically, substances, enzymes, and/or compounds that may degrade RNA, e.g., RNases, are removed from the sample prior to and/or during RNA isolation and/or extraction.
[0072] In some embodiments, total RNA, mRNA, sentinel RNA, tRNA, rRNA, miRNA, and/or other RNA molecules, which may be contained in a biological sample, are isolated. For example, total RNA, in some embodiments, is first isolated from a biological sample using techniques known in the art, such as, e.g., Trizol® (Invitrogen), Ribominus®, or other RNA isolation/extraction kits known in the art. Additionally or alternatively, in some embodiments, subsequently, 5'-capped RNA including, e.g., mRNA and/or sentinel RNA, is separated from the total RNA thereby enriching for the mRNA and/or sentinel RNA, for example. Briefly, the 5 '-capped RNA is isolated from a sample by incubating a tagged cap- binding protein, e.g., eIF-4E, with the total RNA and thereafter purifying the bound complex using methods known in the art, e.g., magnetic or affinity bead isolation and/or chromatography. See, e.g., Folkers et al, "ENCODE tiling array analysis identifies differentially expressed annotated and novel 5 '-capped RNAs in hepatitis C infected liver." PLoS One, 6, el 4697 (2011). Additionally or alternatively, in some embodiments, a high affinity variant of the cap binding protein is employed to enrich for 5 '-cap-isolated RNA. See Choi et al. (2003).
[0073] In this way, 5'-capped RNA can be isolated from other RNAs in the biological sample to enrich for mRNA and/or sentinel RNA, such that the mRNA and/or sentinel RNA is substantially pure, meaning it is at least about 70%, 75%, 80%, 85%, 90%, 95% pure or more, but typically less than 100%) pure, with respect to other RNA molecules. Various kits for the extraction and/or full or partial isolation and/or purification of total RNA, mRNA, and/or one or more sentinel RNAs, from biological samples are provided herein and/or commercially available and are well known to the skilled artisan.
IV. Methods for Identifying sentinel RNAs
[0074] As described herein, progressive 3 '-5' degradation was observed in many of the mRNA transcripts that were evaluated, which therefore indicates that exosome-mediated decay represents one of the primary pathways for mRNA degradation in biological cells. See, e.g., Chen et al., "AU binding proteins recruit the exosome to degrade ARE-containing mRNAs." Cell, 107, 451-464 (2001). Exosome-mediated decay occurs by 3 -5'
exonuclease digestion of mRNA transcripts after deadenylation, i.e., poly(A) shortening, which can be stimulated by AU-rich elements (AREs), growth factors, lymphokines, cytokines, and proto-oncogene mRNA transcripts. See id.
[0075] Decapping of mRNA transcripts, by Dcp2 or related enzymes, and the concomitant 5 '-3' decay by Xrnl, primarily occur subsequent to 3'-end degradation. Moreover, results indicate that total RNA depleted of rRNA showed the same pattern of preferential 3'-end decay (data not shown). Therefore, by analyzing 5'-capped RNA, as compared to poly(A) isolated RNA, the methods of the present technology provide for the identification of previously unidentified, differentially expressed, genes in biological samples. Such novel, unannotated R As, including RNAs possessing short or no 3' poly(A) ends, can be employed as rapidly degrading sentinel RNAs, in some aspects of the present technology.
A. General
[0076] In some embodiments, sentinel RNAs are identified by (A) isolating 5' capped mRNA (e.g., as described above in section III.B), and (B) analyzing these RNAs to determine sequence identity, abundance, and/or sequence integrity (e.g., degradation patterns). For example, candidate sentinel RNAs can be identified by nucleic acid sequencing, in conjunction with bioinforomatics programs and publicly available gene expression databases, such as, e.g., GEO Dataset-GSE5364, which contain genetic profiles from a variety of diseased and corresponding healthy samples, and/or databases such as, for example, the Ensembl human genome database, to identify the candidate sentinel RNA sequences. Additionally or alternatively, bioinformatics programs can be used to determine the relative abundance and/or integrity (e.g., degradation patterns) of the candidate sentinel RNAs.
B. Sequencing
[0077] Nucleic acid sequencing allows for the identification of candidate sentinel RNAs the subsequent evaluation of abundance and degradation patterns of these RNAs. RNA and/or DNA sequencing can be performed using methods known in the art, such as, for example, dideoxy chain termination method of Sanger et al. , Proceedings of the National Academy of Sciences USA, 74, 5463-5467 (1977), with modifications by Zimmermann et al, Nucleic Acids Res., 18: 1067 (1990). Sequencing by dideoxy chain termination method can be performed using Thermo Sequenase (Amersham Pharmacia, Piscataway, NJ), Sequenase reagents from US Biochemicals or Sequatherm sequencing kit (Epicenter Technologies, Madison, Wis.). Sequencing may also be carried out by the "RR
dRhodamine Terminator Cycle Sequencing Kit" from PE Applied Biosystems (product no. 403044, Weiterstadt, Germany), Taq DyeDeoxy™ Terminator Cycle Sequencing kit and method (Perkin-Elmer/ Applied Biosystems) in two directions using an Applied Biosystems Model 373 A DNA or in the presence of dye terminators CEQ™ Dye Terminator Cycle Sequencing Kit, (Beckman 608000). Additionally or alternatively, sequencing can be performed by a method known as Pyrosequencing (Pyrosequencing, Westborough, Mass.). Detailed protocols for Pyrosequencing can be found in: Alderborn et al., Genome Res. (2000), 10: 1249-1265. R A or DNA sequencing can be performed using hybridization techniques, such as, but not limited to, heteroduplex tracking assays, line probe assays, nucleic acid arrays, nucleic acid arrays (DNA chips), bead arrays, and the like. See U.S. Pat. No. 6,300,063 and U.S. Pat. No. 5,837,832.
[0078] Massively parallel sequencing, next generation sequencing, and/or deep sequencing are also be used in the methods described herein. See Voelkerding et al. (2009) Clin. Chem. 55(4):641-658; ten Bosch et al. (2008) J. Mol. Diag. 10(6):484-492;
Harismendy et al. (2009) Genome Biol. 10:R32; de Hoon et al. (2008) Biotechniques 44:627-632). Commercially available systems that use clonally amplified DNA include the Genome Sequence FLX systems from 454 Corporation (a Roche Company), the Genome Analyzer from Illumina, and the SOLiD system from Life Technologies. Single molecule massively parallel sequencers include those from, for example, Helicos Biosciences (the Heliscope) and Pacific Biosciences.
[0079] In some embodiments, next generation RNA sequencing is employed to measure the amount, level, and/or presence of one or more candidate sentinel RNAs. In brief, Pol II RNA transcripts are analyzed in some embodiments by RNA sequencing using, e.g., an Illumina Genome Analyzer 2 (GA2) and standard protocols for preparing and sequencing libraries representing RNA samples, as known in the art. In some embodiments, 5'-capped RNAs and/or total RNA is sequenced. Total RNA and/or 5 '-capped RNA- with rRNA removed in some embodiments-can be sequenced using the Ribominus® for RNA-seq kit (Invitrogen). In some embodiments, such RNAs are fragmented, reverse transcribed to cDNA, and adapted to create a cDNA library. Adapters for cDNAs can be amplified via PCR using, e.g., a Cluster Station (Illumina) and sequenced with an Illumina GA2. See Oler et al., "Human RNA polymerase III transcriptomes and relationships to Pol II promoter chromatin and enhancer-binding factors." Nat Struct Mol Biol, 17, 620-628 (2010).
[0080] Nucleic acid sequencing allows for the identification of the candidate sentinel RNA, and provides the information necessary to develop amplification primers which can be used in the methods discussed below. C. Bioinformatic Interrogation of RNA Sequences to Determine Identity, Abundance and Degradation Patterns
[0081] Bioinformatic analyses of candidate sentinel R As are performed in some embodiments to identify transcripts of interest, i.e., sentinel RNAs. For example, in some embodiments, candidate sentinel RNA sequences are aligned with sequences from one or more databases, such as, but not limited to, for example, the NCBI human genome database, NCBI 37.3 of the human genome {e.g., using the Bowtie aligner), the UCSC Genome Browser, MXSCARNA, Murlet, RNAmine, SCARNA, PHMMTS, PSTAG, Rfold, Stem Kernels, CentroidAlifold, CentroidHomfold, CentroidAlign, miRRim, SCARNA LM, npbfold, RactIP, IPknot, Raccess, Fdur, Rentropy, Rchange GEO Datasets, and/or the Ensembl human genome database, and the like. Such bioinformatic programs can then be employed to identify the candidate sentinel RNA sequences, and/or score genes for abundance, differential expression and/or identify degradation patterns.
[0082] In some embodiments, from about 100; 1,000; 10,000; 100,000; 1,000,000;
10,000,000 to about from 1,000,000; 10,000,000 100,000,000; or 1,000,000,000 RNA sequence Reads Per Kilobase of gene per Million reads ("RPKM" or "sequence reads") are obtained and/or performed. Additionally or alternatively, in some embodiments, from about 1,000,000 to about from 10,000,000 RPKM are obtained and/or performed. Additionally or alternatively, in some embodiments 1, 2, 5, 10, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 RPKM are obtained and/or performed.
[0083] Additionally or alternatively, in some embodiments, RPKM from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or 40 nucleotides to about 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, or 500 nucleotides are aligned to, for example, NCBI 37.3 of the human genome, using the Bowtie aligner. See Oler et al., "Human RNA polymerase III transcriptomes and relationships to Pol II promoter chromatin and enhancer-binding factors." Nat. Struct. Mol. Biol, 17, 620-628 (2010). Additionally or alternatively, in some embodiments, about 30-50 nucleotides are aligned. Id. Alignments can be filtered and selected based on score ranges for example, based on the number of base mismatches (e.g., between 0 and 3 mismatches). Non-limiting applications such as, for example, "USeqDefmedRegionScanSeqs" can be employed to score genes for relative differential expression. By way of example, but not by way of limitation, Useq DefinedRegionsScanSeqs (DRSS) determines the genie sequencing reads of a given gene by summing the reads originating from each exon of that gene. For example, if a gene has three exons then DRSS uses the genomic coordinates for each of those three exons to calculate the number of reads from each exon region and sums those reads for that gene. See also, e.g., Nix et ah, "Empirical methods for controlling false positives and estimating confidence in ChlP-Seq peaks." BMC Bioinformatics, 9, 523 (2008).
[0084] In some embodiments, abundant expression is determined using one or more publicly available gene expression datasets {e.g., GEO Dataset, containing gene profiles from many solid cancers, GSE5364, Ensembl, etc.) and/or other bioinformatic programs as known in the art. In some embodiments, identified sequences {e.g., candidate sentinel RNAs) are defined as abundantly expressed if the signal threshold is approximately two orders of magnitude above background. In some embodiments, background levels for read counts of RNA sequencing data are considered to fall between 0.1 and 1 RPKM. The deeper the sequencing depth the more confidence exists in lower background level cutoffs. An RPKM between 0.3-0.4 has been published recently {see e.g., Blood. 2011;118:el01-l 1; PMID: 21596849; Mol Cell. 2010;40:939-53; PMID: 21172659). Additionally or alternatively, in some embodiments, abundant expression is defined as three, four, five, six, seven, eight, nine or ten orders of magnitude above background.
[0085] Additionally or alternatively, in some embodiments, candidate sentinel RNAs are selected from all genes in the Ensembl human genome. In some embodiments, criteria used to identify candidate sentinel RNAs, include, for example, expressed genes with about 1, 5, 10, 20, 30, 40, 50, 100, 500, 1000 or more RPKMs. Additionally or alternatively, in some embodiments, candidate sentinel RNAs are selected from samples containing expressed genes with an RPKMs of about > 10. Additionally or alternatively, in some embodiments, candidate sentinel RNAs are selected from samples containing expressed genes with a RPKM of one log above background.
[0086] Additionally or alternatively, in some embodiments, abundantly expressed sequences are selected based on 5'- and 3'-end transcription analysis. In this respect, sentinel RNAs can include, but are not limited to abundantly expressed genes with about 1 , 5, 10, 20, 30, 40, 50, 100, 500, 1000 or more RPKMs from the 5'- and/or 3'-terminal ends. Additionally or alternatively, in some embodiments, sentinel RNAs contain abundantly expressed genes with about 10-50 or more RPKMs in the 5'- and/or 3'-terminal ends.
[0087] As described above, additionally or alternatively, in some embodiments, sequencing analyses of sentinel RNAs are employed to interrogate the 5' and 3'-ends, separately, simultaneously, or sequentially. In this respect, in some embodiments, from about 10, 20, 30, 40, 50, 100, 200, 300, 500, 1000, or 10,000 bases to about 50, 100, 200, 300, 500, 1 ,000; 10,000; or 100,000 bases from the 5*- and/or 3*-ends of one or more sentinel RNAs are analyzed. Additionally or alternatively, in some embodiments, from about 100 to about 300 bases from the 5' and/or 3'-ends of one or more sentinel RNAs are analyzed. Additionally or alternatively, in some embodiments, about 200 bases from the 5' and/or 3'-ends of one or more sentinel RNAs are analyzed.
[0088] Additionally or alternatively, in some embodiments, sentinel RNAs possess decreasing or increasing 375' and/or 573 ' ratios-at increasing time intervals-relative to static RNAs and/or RNAs which do not possess such ratios. Additionally or alternatively, in some embodiments, sentinel RNAs possess decreasing 375' ratios as time increases.
Additionally or alternatively, in some embodiments, sentinel RNAs processively decay with 3'-5' and/or 5 '-3 ' polarity. Additionally or alternatively, in some embodiments, candidate sentinel RNAs processively decay with 3 '-5' polarity. Additionally or alternatively, in some embodiments, sentinel RNAs processively decay with 3'-5' polarity at a rapid rate.
Additionally or alternatively, in some embodiments, a rapid decay rate is considered a > 20% decrease in sequencing reads on the 3 ' end (200 terminal nucleotides of the 3 ' untranslated end or UTR) compared to the 5 ' end of sentinel RNAs after 5 and 10 minutes at degradation (room temperature in the examples provided). Additionally or alternatively, in some embodiments, a rapid decay rate is defined as a slope of 0.2 between 0 and 10 minutes of degradation.
[0089] Additionally or alternatively, in some embodiments, RPKM measurements for identifying sentinel RNAs, are selected from sequencing reactions performed after 1 , 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 75, 100, 200, 500, 1000 or more minutes (min) of sample incubation. Additionally or alternatively, in some embodiments, RPKM measurements for identifying sentinel RNAs are selected from sequencing reactions performed after 5, 10, and/or 15 minutes of sample incubation times. Additionally or alternatively, in some embodiments, the samples are incubated at about from 0°C, 4°C, 5°C, 10°C, 15°C, 20°C, 25°C, and/or 37°C to about from 4°C, 5°C, 10°C, 15°C, 20°C, 25°C, 35°C, 50°C and/or 100°C and evaluated at specific time points (e.g., such as those described above). In some embodiments, the samples are incubated at room temperature and evaluated at specific time points (e.g., such as those described above). [0090] Additionally or alternatively, in some embodiments sentinel RNAs are identified via degradation pattern analysis of candidate sentinel RNAs. In some embodiments, to determine such degradation patterns, 5' capped RNA sequences are compared to sequences provided in one or more database(s), using e.g., bioinformatics programs, to identify those RNAs which are (1) abundantly expressed and (2) show rapid degradation relative to the other 5' capped RNAs. In some embodiments, candidate sentinel RNA degradation patterns are determined by calculating the ratio of the number of sequence reads in a defined number of 3' and/or 5 '-end nucleotides. Additionally or alternatively, in some embodiments, the number of sequence reads in a defined number of 3' and/or 5' end nucleotides is determined at different time points to establish a 3 ' end/5 '-end ratio.
[0091] The defined number of nucleotides in the 3' and 5 '-ends can be the same or different. Thus, in some embodiments, the defined number of nucleotides ranges from about 5-1000, about 10-500, about 20-250, and about 30-100. Additionally or alternatively, in some embodiments, the defined number of nucleotides is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or is about 300 or more. Additionally or alternatively, in some embodiments, the defined number of nucleotides of the 3' and/or 5'-end is 200.
[0092] Additionally or alternatively, in some embodiments, candidate sentinel RNA decay (degradation) is measured at different time points, e.g., from the time a sample is collected and/or different time points from when a sample is thawed. In some embodiments, samples are evaluated for degradation at from about 0.1, 0.5, 1, 2, 3, 4, 5 or 6 seconds, minutes, hours, days or years to about from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50 or 100 seconds, minutes, hours, days or years. In some embodiments, sentinel RNA decay is measured at about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 30 minutes, 60 minutes, or 90 minutes or more after sample collection or after sample thaw. Additionally or alternatively, in some embodiments, sentinel RNA decay is measured at time points which simulate sample treatment prior to a biological assay. [0093] In a non-limiting exemplary embodiment, candidate sentinel RNAs are identified as follows. Biological samples continaing RNA are sequenced at TO, Tl, T2, and T3 of incubation at a given temperature. Sequencing is performed such that each sample has given number of sequence reads (e.g., 1-10 million sequence reads) for a fixed number of nucleotides (e.g., 10-50) nucleotides. Sequences are evaluated as follows.
• All genes are identified (e.g, using the gene sequences in the Ensembl human
genome 19 March 2009 build);
• Expressed genes are defined (e.g., those sequences with 10 or more sequencing reads in the T3 sample);
• Abundantly expressed genes are identified (e.g., those sequences with 50 or more reads in the T3 sample);
• Abundantly expressed genes with significant 5' and 3' transcription are identified (e.g., those sequences with 25 or more reads in the terminal 200 bp of their 5' and 3' ends at TO and 25 or more reads in their 5' end after Tl, T2 and T3);
• RNAs with an overall decrease in 375' ratios are identified: (e.g., those RNAs with a slope for the best fit linear line of < -0.2).
• RNAs with progressive 3' to 5' degradation are identified (e.g., those RNAs that show a difference in 375' ratio of > 0.2 between TO, Tl and T2 time points).
V. Methods and Assays for Measuring Biological Sample Integrity
[0094] In some aspects, the amount or level of one or more sentinel RNAs present in a biological sample correlates with the biological integrity of the sample, which may subsequently be used in a diagnostic assay. Accordingly, in some aspects, the present methods can be used to measure sentinel RNA levels or amounts in order to facilitate disease diagnosis and/or therapeutic administration.
[0095] In some embodiments, the methods provided herein can be applied to quantify the amount of sentinel RNA decay, and concomitant intactness, in a biological sample at one or more time points. Embodiments of the present technology include methods for determining the amount of one or more sentinel RNA sequences present in a biological sample, and/or degradation thereof, and comparing the result to a reference standard, such as, e.g., a standard curve, normal or healthy control samples, etc. The difference in the level or amount of one or more sentinel RNA sequences in the biological sample, compared to the reference standard, is indicative of the biological sample quality, which may be
subsequently used for further pathological analysis.
[0096] In some embodiments, sentinel R A levels are determined in a biological sample by amplification.
A. Control or Reference Standards
[0097] As described previously, the sentinel R As may be biomarkers which pertain to a subject and/or patient and can be used to measure the quality or integrity of a sample, and/or the progressive decrease in the quality or integrity of a biological sample as a function of time. Thus, in some embodiments, sentinel RNA standards are developed to simulate sample treatment prior to a biological assay (e.g., a MammaPrint™ Oncotype DX™ or ColoPrint™ assay). Standards can be prepared from a separate, comparable biological sample or the same biological sample to be assayed. Additionally or alternatively, standards can be prepared and evaluated in advance of the biological assay, using an aliquot of the biological sample to be assayed or a comparable sample. Additionally or alternatively, controls or standards can be performed and control or standard information can be provided as values or ranges that indicate sample integrity, to be compared to the sentinel RNA degradation value or range of the biological sample.
[0098] Thus, for example, in some embodiments, if the biological sample shows more sentinel RNA degradation than an "acceptable" range or value indicated by the reference standard, the biological sample integrity is suspect and should not be used in the bioassay, or if used in the assay, results derived from the suspect biological sample should be critically analyzed. Likewise, if the biological sample shows sentinel RNA degradation that falls within an "acceptable" range or value indicated by the reference standard, the biological sample integrity is good, and the bioassay should proceed.
[0099] The skilled artisan will understand how to develop a reference standard or a set of reference standards to mimic sample treatment, e.g., the control or reference should undergo one or more of the same or similar sample collection steps, sample preparation steps (e.g.,
RNA isolation or purification), sample storage steps, and/or the final preparation steps of the sample prior to use in the selected assay application (e.g., reagent addition, incubation, etc.). For example, in some embodiments, time, temperature, reagent addition and/or storage of the sample are considered in developing a reference sample(s). For example, in some embodiments, sentinel RNA decay is measured to develop a standard from about 0.1, 0.5, 1, 2, 3, 4, 5 or 6 seconds, minutes, hours, days or years to about from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50 or 100 seconds, minutes, hours, days or years after sample collection and/or after sample thaw (e.g., at RT, or at 4°C). Additionally or alternatively, in some embodiments, sentinel RNA decay is measured after RNA is isolated from the standard sample, to develop the standard. Additionally or alternatively, in some embodiments, RNA is isolated by methods known in the art, including but not limited to, for example, Trizol® kit and/or 5 '-capped mRNA isolation.
[0100] Additionally or alternatively, in some embodiments, the integrity of a biological sample is compared to standard samples containing one or more sentinel RNAs which have been incubated at a given temperature or temperature range (e.g., 0°C, 4°C, 25°C, room temperature (RT), 37°C, etc.). Additionally or alternatively, the incubation of the control sample is at a temperature from about 1, 10, 20, 30 or 40°C to about 20, 30, 40, 50, 60, 70, 80, 90, 100°C or higher. Additionally or alternatively, a standard curve can be generated to facilitate accurate comparative analyses. Furthermore, the integrity of a biological sample can be compared to standard samples containing one or more sentinel RNAs which have been incubated at a selected temperature from about 0.1, 0.5, 1, 2, 3, 4, 5 or 6 seconds, minutes, hours, days or years to about from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50 or 100 seconds, minutes, hours, days or years. Additionally or alternatively, in some embodiments, sentinel RNA decay to develop standards is measured using the same methods as used for determining sentinel RNA decay in the biological sample.
[0101] Additionally or alternatively, one or more standard curves can be developed and used as a standards or controls. For example, in some embodiments, a control sample is collected and subject to RNase treatment, and/or incubation at a particular temperature, with aliquots being evaluated for sentinel RNA degradation at pre-determined time points (e.g., once per 30 seconds, once per minute, every 2 minutes, every 5 minutes, etc.). Additionally or alternatively, in some embodiments, a control sample is collected and subject to RNase treatment at increasing concentrations of RNase, and/or incubation at a particular temperature and/or for a specific time (e.g., once per 30 seconds, once per minute, every 2 minutes, every 5 minutes, etc.).
[0102] Additionally or alternatively, in some embodiments, the control or standard values of sentinel RNA decay are measured using nucleic acid amplification techniques. In some embodiments, a non-limiting example of a control mRNA showing little change in the 375' qPCR ratio with time relative to a sentienl mRNA would be MT-COl (RNA sequencing data and 375' Ratio provided in FIGURE 9).
B. Detection of sentinel RNA - Amplification Based Assays
1. General
[0103] In some embodiments, amplification-based assays are used to measure the intactness and/or quantity of sentinel RNAs in a biological sample. As noted above, such assays can be used for control or standard determination and/or for biological sample evaluation. Such assays can rapidly assess RNA integrity in biospecimens prior to downstream gene expression analysis or diagnostic/prognostic testing. In some
embodiments, by measuring the quantity of 3 '-end exon regions, for example, as compared to the quantity of 5 '-end exon regions, the respective intactness and quantity of the one or more sentinel RNAs in the biological sample can be determined. Additionally or alternatively, in some embodiments, the intactness of sentinel RNA is determined independent of changes in RNA abundance.
[0104] In this regard, in some embodiments, RNA from a biological sample is isolated as described above. Additionally or alternatively in some embodiments, RNA from a biological sample is isolated using the 5 '-capped RNA isolation procedures described herein and/or total RNA is used. Additionally or alternatively, in some embodiments, a quantitative assessment of sentinel RNA present in a sample is determined. Additionally or alternatively, in some embodiments, in concert with this assessment, the 3 '-end degradation provides data required for determining whether the one or more sentinel RNAs are intact. In some embodiments, the amount of intactness, e.g., non-degraded sentinel RNA, is about from 1, 5, 10, 20, 30, 40, or 50% to about 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more. Conversely, in some embodiments, the amount of sentinel RNA degradation is about from 1, 5, 10, 20, 30, 40, or 50% to about 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more. 2. Degradation Patterns
[0105] Additionally or alternatively, amplification-based procedures can also be performed to assess the ratio of 375' and/or 573' sentinel RNA in a biological sample. In some embodiments, such ratios are measured at a single time point or at multiple, different time points. Embodiments of the present technology include, for example, measuring 375' and/or 573' sentinel RNA degradation ratios in a biological sample at a single time.
Additionally or alternatively, in some embodiments, sentinel RNA of a biological sample is measured at various time points, wherein the time points correlate to treatment of the sample, incubation at a specific temperature, etc., as described above. In some
embodiments, sentinel RNAs in a biological sample possess decreasing 375' ratios as time increases. Additionally or alternatively, in some embodiments, sentinel RNAs processively decay with 3 '-5 polarity. Additionally or alternatively, in some embodiments, sentinel RNAs processively decay with 5 ' - 3 ' polarity. Additionally or alternatively , in some embodiments, sentinel RNAs processively decay in some embodiments with 3 '-5' polarity at a rapid rate. In some embodiments, a rapid decay rate is considered a > 20% decrease in sequencing reads on the 3' end (200 terminal nucleotides of the 3' untranslated end or UTR) compared to the 5' end of sentinel RNAs after 5 and 10 minutes at degradation (room temperature in the examples provided). Additionally or alternatively, in some
embodiments, rapid decay rate is defined as a slope of 0.2 between 0 and 10 minutes of degradation. The foregoing embodiments can be measured, for example, using one or more amplification procedures known in the art.
3. Amplification Methods
[0106] RNA and/or DNA, including, but not limited to complementary forms thereof, can be amplified using nucleic acid amplification techniques well known in the art. By way of example, such techniques include, but are nor limited to, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative reverse transcriptase polymerase chain reaction PCR (qRT-PCR), and/or ligase chain reaction. See Abravaya, K., et al., Nucleic Acids Research, 23:675-682, (1995), branched DNA signal amplification, Urdea, M. S., et al., AIDS, 7 (suppl 2):S11-S 14, (1993), amplifiable RNA reporters, Q-beta replication, transcription-based amplification, boomerang DNA amplification, strand displacement activation, cycling probe technology, isothermal nucleic acid sequence based amplification (NASBA). See Kievits, T. et al, J Virological Methods, 35:273-286, (1991), Invader Technology, or other sequence replication assays or signal amplification assays may also be used. Some of these methods of amplification are described briefly below and are well-known in the art.
[0107] In some embodiments, PCR is used to amplify a sequence of interest, i.e., a sentinel RNA sequence. PCR is a technique for making many copies of a specific template DNA and/or cDNA sequence. In some embodiments, the reaction can include multiple amplification cycles and is initiated using primer sequences that hybridize to the 5 ' and 3 ' ends of the sequence to be copied. In some embodiments, the amplification cycle includes an initial denaturation, and typically up to 50 cycles of annealing, strand elongation and strand separation (denaturation). In some embodiments, in each cycle of the reaction, the DNA and/or cDNA sequence between the primers is copied. In some embodiments, primers can bind to the copied DNA and/or cDNA as well as the original template sequence, so the total number of copies increases exponentially with time. In some embodiments, PCR is performed as according to Whelan et al., J of Clin Micro, 33(3):556- 561 (1995). Briefly, a PCR reaction mixture includes two specific primers, dNTPs, approximately 0.25 U of Taq polymerase, and lx PCR Buffer.
[0108] Some methods of the present disclosure employ reverse transcription of RNA to cDNA. The method of reverse transcription and amplification may be performed by previously published or recommended procedures. Various reverse transcriptases may be used, including, but not limited to, MMLV RT, RNase H mutants of MMLV RT such as Superscript and Superscript II (Life Technologies, GIBCO BRL, Gaithersburg, Md.), AMV RT, and thermostable reverse transcriptase from Thermus thermophilus . For example, one method which may be used to convert RNA to cDNA is the protocol adapted from the Superscript II Preamplification system (Life Technologies, GIBCO BRL, Gaithersburg, Md.; catalog no. 18089-011), as described by Rashtchian, A., PCR Methods Applic, 4:S83- S91, (1994). In some embodiments, quantitative reverse transcription PCR (qPCR) is performed. The skilled artisan will readily understand the various techniques known in the art for performing and modifying qRT-PCR, as described herein.
[0109] The methods may include amplifying multiple nucleic acids in sample, also known as "multiplex detection" or "multiplexing." As used herein, the term "multiplex PCR" refers to PCR, which involves adding more than one set of PCR primers to the reaction in order to detect and quantify multiple nucleic acids, including nucleic acids from one or more target gene markers. Furthermore, multiplexing with an internal control, (e.g., 18s rRNA, GADPH, or β-actin) provides a control for the PCR reaction.
4. Amplification Primers
[0110] Primers can be developed for the amplification and/or quantification assays described herein, for example, using methods well known in the art, e.g., Roche's Universal Probe Library Assay Design Center (Basel, Switzerland). The skilled artisan is capable of designing and preparing primers that are appropriate for amplifying a target or marker sequence. The length of the amplification primers depends on several factors including the nucleotide sequence identity and the temperature at which these nucleic acids are hybridized or used during in vitro nucleic acid amplification. The considerations necessary to determine a preferred length for an amplification primer of a particular sequence identity are well-known to a person of ordinary skill. For example, the length of a short nucleic acid or oligonucleotide can relate to its hybridization specificity or selectivity.
[0111] In some embodiments, primers for detecting one or more sentinel RNA primers may be designed based on the cDNA sequence available for one or more sentinel RNAs including, but are not limited to, e.g., one or more of the following genes or gene products: EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orO, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT, SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, ALB, CLNS1A, ARF1, NDUFV1, RPL10A, SDC4, LASP1, ECHS1, C19orf63, FGG, TNFRSF1A, EIF4EBP1, CDC42EP1, RPN1, HLA-B, TFR2, SERPINA3, TMEM111, TMSB4X, AC025165.27, AMBP, ACAT1, SEC61A1, BRP44L, CTSB, MARS, PCK1, DHCR24, KNG1, TUBA IB, SLC27A5, UBA1, FLU, RHOA, HNF4A, CTSD, IGFBP4, P4HB, LRRC59, SERPINA10, GC, LRPAP1, RPS5, DNAJC3, C4BPB, VTN, IGFBPl, RPN2, BCL2L1, TUBB6, LY6E, FLOT2, NANS, HPN, SERPINF2, SLC9A3R1, ARMET, IDH2, PSMC4, LAPTM4A, GPI, FURIN, APOB, APOH, XBP1, SERPINA11, GUSB, SEMA4B, HPD, KRT18, TMEM176A, RPS2, LRP1, TGM2, FKBP11, TRIB1, SOD2, AC087521.10, BRI3, GABARAP, CRP, YWHAE, GAPDH, SERPINC1, ITIH1, HMOX1, CHI3L1, C14orf68, HPX, TPT1, HSP90B1,
SERPINF1, H19, HSD17B10, ARHGDIA, GDI1, ACTB, KRT8, CFL1, AF235103.4, HABP2, PLA2G2A, ATP1A1, PRDX2, RPS3, DAD1, METTL7B, AXUD1, PSMD13, HM13, C19orfl0, MORF4L2, FNDC4, SRPR, MSN, SERPINB1, RPS4X, SAT1,
TAGLN2, PSMB7, LAMP1, SERPINA1, CPS1, HSPE1, PSME1, TMEM205, TUBB2C, C8B, RPS13, MLF2, RPL4, PMPCA, UQCRC1, LTBR, PEBP1, APOL1, ATP6V0E, TYMP, RPS9, FTL, AQP3, SERPINA7, LRGl, GSTKl, ETFB, GRINA, FGA, C21orf33, TCF25, EIF3K, CSTB, C5, CHAC1, EIF3G, IFITM1, APOA4, VPS28, POLD4, F2, ITGA5, DDX17, HSD17B2, SAT2, FGB, PDIA4, ACADVL, IGFBP2, EIF5A, TMBIM6, OAF, ACY1, PHB2, HLA-E, RHOD, ACOl 1498.7, CIB1, RPL12, GRHPR, RARRES2, TTR, GPX3, CD74, RPL30, HRG, RASD1, C9, SSR2, SHC1, C3, AZGP1, RRBP1, CALR, NDUFA4, GNB2L1, C19orf43, MDH2, GSTOl, LMAN1, APCS, PSMD4, SDS, PPIB, AP001453.6, BLOC1S1, RPL35, GADD45B, RPLP1, TMED9, AARS, SDF2L1, EPN1, PFNl, DUFS6, NDUFB9, GLTPD2, PSMB4, GCHFR, SPCS3, ID1, RABAC1, RTKN, COX8A, PSMC5, GLTSCR2, SF3B5, RPS20, NDUFB2, NEDD8, JUNB, MYL6, EEF2, ARL5B, TOMM7, FAM96B, SAA2, RPLl 1, TRAPPCl, EDFl, CI lorflO, ATP5D, RPL35A, GDI2, SURF4, RPL31, G0S2, SEC61G, NOLA3, SAA4, VAMP 8, IFITM3, C8G, and/or RPL37A, and the like.
[0112] In some embodiments, the one or more sentinel RNA primers are provided.
Exemplary, non-limiting primers are provided in the table below. In some embodiments, the primers are for one or both of GNB2L1 and TMBIM6.
Figure imgf000042_0001
GENE
5' end ggccatggcgctgctact aggcagctcgagcccagt
3' end atatgggtagggggaggtgt accccataccccttattgct
GNB2L1
5' end acgaagggtcatctgctca ctaagccatccagtgccatc
3' end atggtggggacatcatcaac gcagcacacagccagtagc
TMBIM6
5' end atatggtcactcatttcattcagg tcaatatcagggagcccaag
3' end catccagcctttcccaatta gtgtgcaaaaaggaaatgagg
MYL6
5' end aagaccagaccgcagagttc ggatcttgccatcacctgtt
3' end agaggaagaagtagagatgctggt tgcctcacaaacgcttcata
JUNB
5' end atacacagctacgggatacgg gctcggtttcaggagtttgt
3' end caaggtgaagacgctcaagg tea tga ccttctgtttgagctg
SAA2
5' end aaaagcctcgccaaggaa ttctgctccttggtcctga
3' end ccagcaggtcggaagtga ccgatcaggctgccaata
TIMP1
5' end tgttgttgctgtggctgatag aggteggaattgeagaagg
3' end tatccatcccctgcaaactg gactggaagcccttttcaga
Slow Degrader
MT-BCOl
5' end gcttctga ctctta cctccctct ccggcctccactatagca
3' end tgtagctcacttccactatgtcct aagcctcctatgatggcaaa
[0113] The amplification reactions on the present technology may include a labeled primer or probe, thereby allowing detection of the amplification products corresponding to that primer or probe. In one embodiment, the amplification may include a multiplicity of labeled primers or probes; such primers may be distinguishably labeled, allowing the simultaneous detection of multiple amplification products. In one embodiment, a primer or probe is labeled with a fluorogenic reporter dye that emits a detectable signal. While a suitable reporter dye is a fluorescent dye, any reporter dye that can be attached to a detection reagent such as an oligonucleotide probe or primer is suitable for use in the invention. Such dyes include, but are not limited to, Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Edans, Eosin, Erythrosin, Fluorescein, 6-Fam, Tet, Joe, Hex, Oregon Green, Rhodamine, Rhodol Green, Tamra, Rox, and Texas Red.
5. Amplicon Detection
[0114] Additionally or alternatively, in some embodiments, amplification is monitored using "real-time" methods. Real time PCR allows for the detection and quantitation of a nucleic acid target. Typically, this approach to quantitative PCR utilizes a fluorescent dye, which may be a double-strand specific dye, such as SYBR Green® I. Alternatively, other fluorescent dyes, e.g., FAM or HEX, may be conjugated to an oligonucleotide probe or a primer. Various instruments capable of performing real time PCR are known in the art and include, for example, ABI Prism® 7900 (Applied Biosystems) and LightCycler® systems (Roche). The fluorescent signal generated at each cycle of PCR is proportional to the amount of PCR product. A plot of fluorescence versus cycle number is used to describe the kinetics of amplification and a fluorescence threshold level is used to define a fractional cycle number related to initial template concentration. When amplification is performed and detected on an instrument capable of reading fluorescence during thermal cycling, the intended PCR product from non-specific PCR products can be differentiated using melting analysis. By measuring the change in fluorescence while gradually increasing the temperature of the reaction subsequent to amplification and signal generation it may be possible to determine the Tm of the intended product(s) as well as that of the nonspecific product.
[0115] While some methods described herein also relate to PCR amplification in general, numerous other methods are known in the art for enzymatic amplification and reproduction of nucleic acids. For example, other enzymatic replication and amplification methods include, but are not limited to, isothermal methods, rolling circle methods, Hot-start PCR, real-time PCR, Allele-specific PCR, Assembly PCR or Polymerase Cycling Assembly (PCA), Asymmetric PCR, Colony PCR, Emulsion PCR, Fast PCR, Real-Time PCR, nucleic acid ligation, Gap Ligation Chain Reaction (Gap LCR), Ligation-mediated PCR,
Multiplex Ligation-dependent Probe Amplification, (MLPA), Gap Extension Ligation PCR (GEXL-PCR), quantitative PCR (Q-PCR), Quantitative real-time PCR (QRT-PCR), multiplex PCR, Helicase-dependent amplification, Intersequence-specific (ISSR) PCR, Inverse PCR, Linear-After-The-Exponential-PCR (LATE-PCR), Methylation-specific PCR (MSP), Nested PCR, Overlap-extension PCR, PAN- AC assay, Reverse Transcription PCR (RT-PCR), Rapid Amplification of cDNA Ends (RACE PCR), Single molecule
amplification PCR (SMA PCR), Thermal asymmetric interlaced PCR (TAIL-PCR), Touchdown PCR, long PCR, nucleic acid sequencing (including DNA sequencing and RNA sequencing), transcription, reverse transcription, duplication, DNA or RNA ligation, and other nucleic acid extension reactions known in the art.
6. End Ratio Calculations
[0116] Additionally or alternatively, in some embodiments, 375 '-end expression ratios are generated for each test RNA transcript using formulas for quantification in real-time RT- PCR as described. Pfaffl, M.W. "A new mathematical model for relative quantification in real-time qRT-PCR." Nucleic acids research, 29, e45 (2001). In some embodiments, the formula is as follows:
ratio - ^ *xs?&- eF**fsimvte
[0117] In some embodiments, the 375' ratio data equals the efficiency of the target qRT- PCR raised to the power of cycle threshold for the target control subtracted from the cycle threshold of the target sample. Additionally or alternatively, in some embodiments, the ascertained value is subsequently divided by the efficiency of the reference raised to the cycle threshold of the reference control subtracted from the cycle threshold of the reference sample. Additionally or alternatively, in some embodiments, an efficiency of two is employed for all calculations. In some embodiments, exact PCR efficiencies (generally between 1.8 and 2.2) may be calculated for both 5 ' and 3 ' end primer pairs to most accurately calculate 375' ratios. Additionally or alternatively, in some embodiments, the above formula target and reference are defined as the 3'- and 5 '-end amplicons of the same sentinel RNA, respectively. Cycle thresholds and the 0 minute time point can serve as the cycle threshold control in some embodiments.
7. Assay Panels [0118] Additionally or alternatively, in some embodiments, the sentinel RNAs described herein, as listed above, can be grouped in non- limiting assay panels for use in the 375' qRT- PCR assays methods described herein. In some embodiments, at least from about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, or 500 sentinel RNAs to about 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 1 ,000, or 10,000 sentinel RNAs are employed for the 3 5' qRT-PCR assays in accordance with the methods provided herein.
[0119] Additionally or alternatively, in some embodiments, the 375' qRT-PCR assays described herein are analyzed in comparison to reference standards and/or control samples, from which standard curves were generated for specific tissues and/or assays, as described above. Additionally or alternatively, in some embodiments, the minimum number of sentinel RNAs selected for a 375' qRT-PCR assay to detect degradation of about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, or 90% of the niRNAs in a biological sample are be determined. Additionally or alternatively, in some embodiments, the minimum number of sentinel RNAs selected is about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, or 90% of the mRNAs in a biological sample. Additionally or alternatively, in some embodiments, the minimum number of sentinel RNAs selected is about 5% of the mRNAs in a biological sample.
[0120] In some embodiments, >3 sentinel RNAs are used to determine the RNA integrity of a biospecimens sample. In some embodiments, the exact number of sentinels selected will be determined, in part, by the tissue being sampled as some tissues may have lower overall expression of some sentinels.
[0121] Additionally or alternatively, in some embodiments, the number of sentinel RNAs selected (e.g., for a panel) is about 10. In some embodiments, a panel of sentinel RNAs includes one or more of the following: SERPINA3, B2M, ENOl , GNB2L1 , TMBIM6, PFN1 , MYL6, SAA2, JUNB and TIMP1. 8. Statistical Methods
[0122] Additionally or alternatively, in some embodiments, statistical methods can be used to set thresholds for determining when the expression level in a subject can be considered to be different than or similar to the reference level. As used herein, the phrase "difference of the level" refers to differences in the quantity of a sentinel RNA present in a sample taken from patients having or suspected of having a disease or medical condition as compared to a control. In some embodiments, a sentinel RNA may be present at an elevated amount or at a decreased amount in samples of patients having or suspected of having a disease or medical condition compared to a reference level. In some embodiments, a "difference of a level" may be a statistically significant difference. For example, a difference may be statistically significant if the measured level of the biomarker falls outside of about 1.0 standard deviations, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 stand deviations of the mean of any control or reference group.
[0123] Additionally or alternatively, in some embodiments, statistics can be used to determine the validity of the difference or similarity observed between a patient's gene expression level and the reference level. Exemplary statistical analysis methods are described in L.D. Fisher & G. vanBelle, Biostatistics: A Methodology for the Health Sciences (Wiley-lnterscience, NY, 1993). For instance, confidence ("/?") values can be calculated using an unpaired 2-tailed t test, with a difference between groups deemed significant if the p value is less than or equal to 0.05.
C. Detection of sentinel RNA - Other Methods
[0124] Other methods for measuring the level or amount of one or more sentinel RNAs in a biological sample including, but are not limited to, e.g., hybridization assays using detectably labeled DNA or RNA probes, i.e., Northern blotting, or quantitative or semiquantitative RT-PCR methodologies using appropriate oligonucleotide primers.
Alternatively, quantitative or semi-quantitative in situ hybridization assays can be carried out using, for example, tissue sections, or unlysed cell suspensions, and detectably labeled, e.g., fluorescent, or enzyme-labeled, DNA or RNA probes. Additional methods for quantifying mRNA include RNA protection assay ("RPA"), cDNA and oligonucleotide microarrays, representation difference analysis ("RDA"), differential display, EST sequence analysis, serial analysis of gene expression ("SAGE"), and multiplex ligation-mediated amplification with the Luminex FlexMAP ("LMF"). See Peck et al, Genome Biol., 7(7):R61 (2006).
[0125] The skilled artisan will understand that additional or alternative methods may be used either in place of, or together with, PCR methods, including enzymatic replication reactions developed in the future. See, e.g., Saiki, "Amplification of Genomic DNA" in PCR Protocols, Innis et al, eds., Academic Press, San Diego, CA, 13-20 (1990); Wharam, et al., 29(11) Nucleic Acids Res, E54-E54 (2001); Hafner, et al., 30(4) Biotechniques, 852- 6, 858, 860 passim (2001); Ross, P., et al, International Patent Appl. No. WO 91/06678; Kwiatkowski, M., United States Patent Nos. US 6,255,475, US 6,309,836, and US 6639088 and EP1218391; Anazawa, T., et al., United States Patent No. 6242193; Ju, et al., United States Patent No. US 6,664,079; Tsien, R.Y., et al, International Patent Appl. No. WO 91/06678; and Dower, et al, International Patent Appl. No. WO 92/10587.
VI. Kits
[0126] Reagents employed in the disclosed methods can be packaged into diagnostic kits and the like. Diagnostic kits can include, for example, at least one or more primers specific for one or more sentinel RNAs. Additionally or alternatively, in some embodiments the kit includes nucleotide bases capable of being incorporated into an elongating oligonucleotide by a polymerase. Additionally or alternatively, in some embodiments, the bases are labeled. Additionally or alternatively, in some embodiments, specific labeling reagents can also be included in the kits as disclosed herein. Additionally or alternatively, in some embodiments, the kit can also contain other suitably packaged reagents and materials needed for amplification, for example, buffers, dNTPs, or polymerizing enzymes, and for detection analysis, for example, enzymes and solid phase extractants. Additionally or alternatively, in some embodiments, the kits comprise multiple amplification primer sets, wherein at least one of the primers is composed of a sequence complementary to at least a portion of one or more sentinel RNAs, such as, e.g., GNB2L1 and TMBIM6, or any other sentinel RNA as described herein. Additionally or alternatively, in some embodiments, the kits include controls and/or standards.
[0127] Additionally or alternatively, in some embodiments, kits include one or more of the following (consistent with methods, reagents, and compositions discussed above):
components for sample purification, including a lysis buffer with a chaotropic agent; a glass-fiber filter or column; an elution buffer; a wash buffer; an alcohol solution; and/or a nuclease inhibitor. The components of the kits may be packaged either in aqueous media or in lyophilized form, for example, and will be provided in a suitable container. The components of the kits provided herein may be provided as dried powder(s). Additionally or alternatively, in some embodiments, when reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent.
Additionally or alternatively, in some embodiments, the solvent may also be provided in another container. The container will generally include at least one vial, test tube, flask, bottle, syringe, and/or other container means, into which the solvent is placed, optionally aliquoted. Additionally or alternatively, in some embodiments, the kits also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other solvent.
[0128] Reagents useful for the disclosed methods can be stored in solution or can be lyophilized. When lyophilized, some or all of the reagents can be readily stored in microtiter plate wells for easy use after reconstitution. It is contemplated that any method for lyophilizing reagents known in the art would be suitable for preparing dried down reagents useful for the disclosed methods.
[0129] Additionally or alternatively, in some embodiments, the kits include control samples or standards, and/or control values or standards.
[0130] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.
EXAMPLES
[0131] The present compositions, methods and kits described herein will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present methods and kits.
I. Overview [0132] Described below are methods for identifying and assessing specific mRNAs, designated sentinel RNAs, which represent biomarkers that reflect of the quality of most mRNA contained in a biological sample. Next generation RNA-sequencing was employed as described in the Examples below to analyze 5 '-capped mRNA recovered from human liver biospecimens which were thawed, and subsequently incubated at RT for increasing time periods. In this way, candidate sentinel mRNAs were identified using sequencing data by calculating the RPKM ratio in the 3'- and 5 '-terminal ends of transcripts at varying times.
II. Materials and Methods
[0133] Tissue and Extraction. Samples of unused human donor liver were obtained with IRB approval and flash frozen with liquid nitrogen and stored at -80°C. Tissue samples were allowed to thaw at room temperature for 0, 5, 10, and 15 min before isolating total RNA. Total RNA was isolated using Trizol® (Invitrogen, Carlsbad, CA) following standard methods. See, e.g., Choi et ah, "Purifying mRNAs with a high-affinity eIF4E mutant identifies the short 3' poly(A) end phenotype." Proceedings of the National Academy of Sciences of the United States of America, , 7033-7038 (2003). The quality of total RNA was measured using a bioanalyzer from Agilent Technologies (Santa Clara, CA).
[0134] Isolation of 5 '-capped RNA. RNA polymerase II transcripts possessing a 5' m7GpppN cap were purified using a recombinant high affinity variant of eIF4E
(eIF4EKl 19A) which binds to the 5'-cap with at least a tenfold higher affinity than the wild- type protein. See Spivak-Kroizman et al., "Mutations in the S4-H2 loop of eIF4E which increase the affinity for m7GTP." FEBS Lett, 516, 9-14 (2002); Friedland et al, "A mutant of eukaryotic protein synthesis initiation factor eIF4E(Kl 19A) has an increased binding affinity for both m7G cap analogues and eIF4G peptides." Biochemistry, 44, 4546-4550 (2005). GST-tagged eIF4EKl 19A protein bound to glutathione-agarose beads was used to purify 5'-capped RNA as described. See, e.g., Choi et al. (2003).
[0135] Next Generation RNA Sequencing. RNA polymerase II transcripts were analyzed by RNA sequencing with an Illumina Genome Analyzer 2 (GA2) and using standard protocols for preparing and sequencing libraries representing RNA samples. 5'-capped RNAs with rRNA removed (Ribominus® for RNA-seq kit, Invitrogen, Carlsbad, CA) were fragmented, reverse transcribed to cDNA, and adapters added to create a cDNA library. cDNAs with adapters were amplified by PCR using a Cluster Station (Illumina, San Diego, CA) and sequenced with an Illumina GA2. see Oler et al., "Human R A polymerase III transcriptomes and relationships to Pol II promoter chromatin and enhancer-binding factors." Nat. Struct. Mol. Biol., 17, 620-628 (2010). Approximately ten million sequence reads of 36 nucleotides were obtained from each RNA sample.
[0136] Bioinformatic Analysis of sentinel RNAs. Sequence reads of 36 nucleotides from each liver RNA sample were aligned to the NCBI 37.3 build of the human genome using the Bowtie aligner {see Oler et al. (2010)) and filtered for alignment score based on e.g., the number of base mismatches {e.g., between 0 and 3 mismatches). The
"USeqDefinedRegionScanSeqs" application was employed to score genes for differential expression relative to the 0 min time point. See Nix et al., "Empirical methods for controlling false positives and estimating confidence in ChlP-Seq peaks." BMC
Bioinformatics, 9, 523 (2008). The bioinformatic criteria used to identify candidate sentinel RNAs were as follows: all genes - 36,615 genes in the Ensembl human genome 19 March 2009 build; expressed genes - 12,166 genes with 10 or more sequencing reads in the 15 minute sample; abundantly expressed genes - 4,877 genes with 50 or more reads in the 15 minute sample; abundantly expressed genes with significant 5' and 3' transcription - 706 genes with 25 or more reads in the terminal 200 bp of their 5' and 3' ends at 0 minutes and 25 or more reads in their 5' end after 5, 10, and 15 minutes; RNAs with an overall decrease in 375' ratios - 565 RNAs with a slope for the best fit linear line of < -0.2; and RNAs with progressive 3' to 5' degradation - 304 RNAs showed a difference in 375' ratio of > 0.2 between 0, 5, and 10 minute time points. After ten minutes half of these transcripts (148 or 49%) had < 25 reads in their terminal 200 bp 3' end.
[0137] Quantitative Reverse Transcription PCR. Gene specific primers were developed for the 3' and 5' ends of sentinel and slowly degrading transcripts. Using Roche's Universal
Probe Library Assay Design Center (Basel, Switzerland), appropriate primers and monocolor hydrolysis probes were chosen. Forward and reverse primers were mixed in IX
LightCycler 480 Probes Master mix containing 100 nMol/L of the ULP probe and 50 ng of cDNA as template. The cDNA was produced using random hexamers from two different preparations of RNA: 5 '-cap dependent RNA isolation and total RNA treated with
Ribominus®. PCR was performed in 96-well plates in a final reaction volume of 20 μΐ.
Each reaction was performed in triplicate. Using a formula as shown below and described in Pfaffl, M.W. "A new mathematical model for relative quantification in real-time RT-
PCR." Nucleic acids research, 29, e45 (2001), 375'-end expression ratios were generated for each RNA transcript. The 375' ratios equaled the efficiency of the target qPCR raised to the power of cycle threshold of the target control minus the cycle threshold of the target sample. That value was then divided by the efficiency of a first reference also raised to the cycle threshold of the reference control minus the cycle threshold of the reference sample. An efficiency of two was used for all calculations. Target and reference were defined as the 3' and 5 '-end amplicons of the same sentinel RNA, respectively. Cycle thresholds and the 0 minute time point was the cycle threshold control. The 5, 10, and 15 minute times were the cycle threshold sample unknowns. The ratio formula is as follows.
ratio
Figure imgf000052_0001
III. Experimental Examples
1. Example 1 - Identifying mRNAs with different decay rates by RNA
sequencing analysis of 5' capped RNA
[0138] To determine the relative rates of annotated RNA polymerase II gene mRNA decay, total RNA was isolated from human liver biospecimens, which were initially snap frozen, and then thawed for 0, 5, 10, and 15 min at room temperature. The total RNA obtained from each sample possessed RNA Integrity Number (RIN) values of 9.5, 8.9, 7.9, and 6.7, respectively (see Figure 1). These values fall within the recommended guidelines for intact high quality RNA that would be examined by gene arrays, RNA sequencing (RNA-seq), and in clinical diagnostic or prognostic gene array testing. See, e.g., Schroeder et al., "The RIN: an RNA integrity number for assigning integrity values to RNA
measurements." BMC molecular biology, 7(3) (2003). Total RNA was used to isolate 5'- capped RNA, which was analyzed by RNA sequencing, as described above.
[0139] The sequence reads-Reads Per Kilobase of gene per Million (RPKM)-were analyzed and aligned using the Integrated Genome Browser (IGB) for approximately 36,000 Ensembl genes. Surprisingly, when compared to the time zero control, a > 50% reduction in RPKM was observed in 2.7%, 14%>, and 76%> of the annotated coding transcripts after 5, 10 and 15 min room temperature incubations, respectively (see Figure 2). Moreover, 80% at five minutes, 54% at ten and <10% of protein coding genes at 15 minutes retained greater than 70%) of their sequencing reads (Figure 3).
[0140] Analysis of the RNA-seq data for specific mRNAs showed a dramatic decline in sequence reads at the 3' end of many transcripts - providing evidence supporting the model that the majority of mammalian mRNAs initially undergo a processive 3' to 5' degradation. However, the rates of 3' to 5' degradation varied significantly among transcripts, and stratified many into classes of rapid and slowly degrading transcripts. The aligned sequence reads of representative genes and their mRNA are shown in IGB to portray representative rapidly and slowly degrading transcripts (Figure 4A-4D; Figure 8A-8J). Briefly, the vertical bar represents the total number of reads for a 36 nucleotide sequence of isolated 5'- capped RNA, which was aligned with the corresponding genomic region, as provided (Hg 19, 2009). Rapidly degrading transcripts, such as, e.g., JUNB {see Figure 4B), rapidly decreased in sequence read number from the 3'-end as time increased. In this respect, 33%, 60%) and 78% reductions in JUNB 3' reads were observed after 5, 10 and 15 min, respectively. Likewise, PFN1 decreased by 39%>, 66%>, and 86%> after 5, 10 and 15 min, respectively (Figure 4A). HIST1H1E {see Figure 4C) and MT-BCOl {see Figure 4D) data is also shown.
[0141] To further classify mRNAs degradation, the 3 '-end and 5 '-ends (200 bases from each terminus) from coding transcripts, which provided for at least one sequencing read, were analyzed at all time points. This data subset represented approximately 15% (-3,000 of the 20,000 assayed) of the coding transcripts. Surprisingly, more than 30%> of these transcripts demonstrated > 50% reduction in 3' reads compared to 5' reads after 5 min, while > 70%) of the transcripts possessed a > 50%> reduction of the same after 10 min {see Figure 5). This result was unexpected in view of the corresponding > 7 RIN value, which is considered satisfactory for diagnostic and prognostic testing, from the same sample. See, e.g., Bueno-de-Mesquita et ah, "Use of 70-gene signature to predict prognosis of patients with node-negative breast cancer: a prospective community-based feasibility study
(RASTER)." Lancet Oncol, 8, 1079-1087 (2007). These findings indicate that most mammalian mRNAs degrade in a 3 -5' processive manner, and that a large percentage of mRNAs can be extensively degraded in biospecimens despite acceptable RIN values.
[0142] To further evaluate the present dataset, read counts from ascertainable exonic, intronic, and intergenic sequences were compared {see Figure 6). At each time point, a reduction in exonic reads was observed, whereas between 5 and 15 min a decrease from 2- 9.5% is shown as compared to intronic and intergenic reads (see Figure 6). Both intronic (2-fold) and intergenic (3.6-fold) were increased over the same interval. Without wishing to be bound by theory, this may reflect a more rapid degradation of mature cytosolic mRNAs represented by exonic sequence reads, or the protein "coat" that various mRNAs possess, may facilitate regulation of RNA decay. See Wilusz et ah, "Consequences of mRNA wardrobe malfunctions." Cell, 143, 863-865 (2010). The observed increase in intronic and intergenic reads may reflect the relative increase in ratio of intronic and intergenic RNAs compared to exonic RNAs for the 15 min interval.
2. Example 2 - Identification of 304 Candidate sentinel RNAs
[0143] Candidate sentinel mRNAs were identified as described in Example 1, i.e., by employing RNA sequencing data and calculating the ratio of sequence reads in the last 200 3 '-end bases to the first 200 5 '-end bases (3' end/5' end ratio), at various time points. A total of 304 of candidate sentinel RNAs were identified as shown below in Table 2.
Table 2 Ratio
Figure imgf000055_0001
frame 3
Figure imgf000055_0002
I
Min
Figure imgf000056_0001
Figure imgf000056_0002
Figure imgf000057_0001
Figure imgf000058_0001
15
Figure imgf000059_0001
Figure imgf000059_0002
Figure imgf000060_0001
Ratio
Figure imgf000061_0001
Figure imgf000061_0002
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
375' Ratio
Figure imgf000065_0001
Figure imgf000065_0002
If
Min
Figure imgf000066_0001
Figure imgf000066_0002
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
I
Min
0.32
0.11
0.1
0.03
0.17
0.14
0.2
0.16
0.16
0.13
0.15
0.14
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000071_0002
375' Ratio
Figure imgf000072_0001
Figure imgf000072_0002
Figure imgf000073_0001
stocompat ty
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000075_0002
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000077_0002
Figure imgf000078_0001
Figure imgf000078_0002
Figure imgf000079_0001
Figure imgf000079_0002
Figure imgf000080_0001
Figure imgf000080_0002
-J
oo
3. Example 3 - Quantitative PCR of 3' and 5' ends of Candidate sentinel mRNAs
[0144] Identifying representative, or "sentinel" RNAs, that reflect the integrity of most mRNAs in a sample is useful in developing improved and more rapid measures of RNA integrity in biospecimens. As described in Example 2, 304 candidate sentinel mRNA transcripts were identified that were rapidly degraded in a 3' to 5' processive order, by mining the Pol II RNA-seq dataset described with specific criteria. These RNAs, or variants thereof, can be used to determine the integrity of a biological sample. In some embodiments, a candidate sentinel RNA is defined as a protein coding transcript that has specific expression criteria and shows a difference in 375' sequence reads of >0.2 between 0.5 and 10 minutes (see Materials and Methods). The integrity of the 3' and 5' ends of a subset of these candidate sentinel mRNAs was further examined by 375' quantitative reverse transcriptase PCR (qPCR) analysis. Seven out of ten sequences examined showed a progressive loss of 3' relative to 5' regions based on qPCR assays. For some genes, the design of qPCR primers was constrained by short 5 ' or 3' end exons or sequence similarity to other transcripts resulting in few primer possibilities. Two transcripts, GNB2L1 and TMBIM6, had 375' qPCR assay results verifying a dramatic 3' to 5' processive degradation (Figure 7A, 7B are GNB2L1; Figure 7C, 7D are TMBIM6). At ten minutes, there was a 50% and 46% loss of the 3' end of GNB2L1 and TMBIM6 transcripts, respectively.
[0145] Several transcripts investigated exhibited a classic 3' to 5' processive degradation pattern as measured by qPCR analysis (see e.g., Figures 8A-8J). The time dependent RNA- seq data and chromosome coordinates of the qPCR amplicons for both GNB2L1 and TMBIM6 are shown in Figures 7A and 7C. To investigate whether isolating RNAs by their 5' cap might bias the analysis we also performed RT-qPCR on total RNA that had ribosomal RNA removed with a Ribominus® protocol (Figure 10). Analyzing this RNA with the same qPCR primers we observed a reduced 3 5' ratio with increasing time for B2M, GNB2L1 and TMGIM6 mRNA confirming that these mRNAs were rapidly degrading in a 3' to 5' manner.
[0146] A number of mRNA transcripts showing little or negligible degradation in the RNA-seq dataset many of which were mitochondrial mRNAs suggesting a possible different mechanism for their degradation as compared to cytosol mRNAs. One
mitochondrial transcript, MT-BC01, showed little degradation (<6%) at the longest time (15 min) as measured by 375' qPCR analysis (Figure 9 A and B). HIST1H1E is another mRNA that showed very slow degradation with time (Figure 4C). Such transcripts provide useful controls.
4. Example 4 - Assaying sentinel RNA in a Biological Sample
[0147] qRT-PCR is employed as follows to facilitate the determination of biological sample quality via sentinel RNA analysis. First, a standard curve is prepared using a fresh tissue of interest and incubating at room temperature for 0, 10, 20, 40, 60 minutes. Each time point is divided into three sets thereby allowing for in parallel analysis through standard gene microarrays, such as, e.g., Affymetrix, to measure mRNAs of the most expressed genes, while performing pair-wise differential gene expression analyses. The number of genes that are determined to be differentially expressed are a direct measure of a "false positives" due to, e.g., sample degradation at the thresholds used to select the differentially expressed genes. For example, one skilled in the art might select differentially expressed genes at a false discovery rate ("FDR") of 1% and 2X difference. In some embodiments, the FDR of a sentinel RNA is < 0.1. At these thresholds, differentially expressed genes at the specified time points are assessed. In some embodiments, it is expected that approximately 10, 20, 50, 75, 100, 120, 150 or more genes are differentially expressed due to degradation. In some embodiments, threshold stringency is increased to accommodate acceptable FDR.
[0148] Subsequently, qRT-PCR is performed on tissue specific sentinel RNAs at each time point. Initially, this is performed at each time point with an unknown RNA set. The 375' ratio is calculated for each gene, at each time point, after converting the 3 '-end terminus and 5 '-end terminus cycle number to relative abundance. As such, at each time point an array of 375' ratios is obtained. Next the test sample is measured against a standard curve by calculating pair-wise Euclidian distances. The time points most closely associated with the test sample serve to score the sample in minutes of degradation. Acceptable thresholds of RNA integrity are empirically determined for each application, e.g., using a fresh sample of a known quality of RNA, while subjecting such a sample to degradation using RNase and the like, and subsequently assaying the sample. 5. Example 5 - Detection of sentinel RNA Expression, Abundance in Breast, Colon and Cancer Samples
[0149] In addition to the experimental model of human liver, described above, expression of GNB2L1 and TMBIM6 in breast, colon, and multiple solid cancers were evaluated to determine if they are also expressed in these tissues. Using a publicly available gene expression dataset (GEO Dataset, GSE5364) containing gene profiles from many solid cancers and their corresponding normal tissue abundant expression was observed (signal threshold approximately two orders of magnitude above background) of these and the other candidate sentinel mRNAs that were identified in human liver biospecimens. This suggests that some sentinel mRNAs may be used for measuring mRNA degradation in a variety of tissues thus reducing the complexity of a sentinel mRNA panel used to analyze the integrity of mRNA in samples before gene expression studies.
* * * *
[0150] It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
[0151] In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member, any subgroup of members of the Markush group or other group, or the totality of members of the Markush group or other group. [0152] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non- limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as "up to," "at least," "greater than," "less than," and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 nucleotides refers to groups having 1, 2, or 3 nucleotides. Similarly, a group having 1-5 nucleotides refers to groups having 1, 2, 3, 4, or 5 nucleotides, and so forth.

Claims

What is claimed is:
1. A method for determining the integrity of a biological sample, the method
comprising:
(a) identifying one or more sentinel RNAs in the biological sample;
(b) determining the amount of degradation in the one or more sentinel RNAs;
(c) comparing the amount of degradation to a reference standard; and
(d) correlating the degradation in the biological sample to the integrity of the biological sample.
2. The method of claim 1, wherein the biological sample is selected from the group consisting of blood, plasma, serum, lymph, mucus, sputum, tears, urine, stool, saliva, tissue, hair, animal cells, and plant cells.
3. The method of any one of the previous claims, wherein the one or more sentinel RNAs are mRNA.
4. The method of any one of the previous claims, wherein the one or more sentinel RNAs are selected from the group consisting of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orf3, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLPO, CRAT, SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, ALB, CLNS1A, ARF1, NDUFV1, RPL10A, SDC4, LASP1, ECHS1, C19orf63, FGG, TNFRSF1A, EIF4EBP1, CDC42EP1, RPN1, HLA-B, TFR2, SERPINA3, TMEMl 11, TMSB4X, AC025165.27, AMBP, ACATl, SEC61A1, BRP44L, CTSB, MARS, PCKl, DHCR24, KNGl, TUBAIB, SLC27A5, UBAl, FLU, RHOA, HNF4A, CTSD, IGFBP4, P4HB, LRRC59, SERPINAIO, GC, LRPAP1, RPS5, DNAJC3, C4BPB, VTN, IGFBP1, RPN2, BCL2L1, TUBB6, LY6E, FLOT2, NANS, HPN, SERPINF2, SLC9A3R1, ARMET, IDH2, PSMC4, LAPTM4A, GPI, FURIN, APOB, APOH, XBP1, SERPINA11, GUSB, SEMA4B, HPD, KRT18, TMEMl 76 A, RPS2, LRP1, TGM2, FKBP11, TRIB1, SOD2, AC087521.10, BRI3, GABARAP, CRP, YWHAE, GAPDH, SERPINC1, ITIH1, HMOX1, CHI3L1, C14orf68, HPX, TPT1, HSP90B1, SERPINF1, H19,
HSD17B10, ARHGDIA, GDI1, ACTB, KRT8, CFL1, AF235103.4, HABP2, PLA2G2A, ATP1A1, PRDX2, RPS3, DAD1, METTL7B, AXUD1, PSMD13, HM13, C19orfl0, MORF4L2, FNDC4, SRPR, MSN, SERPINB1, RPS4X, SAT1, TAGLN2, PSMB7, LAMP1, SERPINA1, CPS1, HSPE1, PSME1, TMEM205, TUBB2C, C8B, RPS13, MLF2, RPL4, PMPCA, UQCRCl, LTBR, PEBPl, APOLl, ATP6V0E, TYMP, RPS9, FTL, AQP3, SERPINA7, LRG1, GSTK1, ETFB, GRINA, FGA, C21or03, TCF25, EIF3K, CSTB, C5, CHAC1, EIF3G, IFITM1, APOA4, VPS28, POLD4, F2, ITGA5, DDX17, HSD17B2, SAT2, FGB, PDIA4, ACADVL, IGFBP2, EIF5A, TMBIM6, OAF, ACY1, PHB2, HLA-E, RHOD, ACOl 1498.7, CIB1, RPL12, GRHPR, RARRES2, TTR, GPX3, CD74, RPL30, HRG, RASD1, C9, SSR2, SHC1, C3, AZGP1, RRBP1, CALR, NDUFA4,
GNB2L1, C19orf43, MDH2, GSTOl, LMAN1, APCS, PSMD4, SDS, PPIB, AP001453.6, BLOC1S1, RPL35, GADD45B, RPLP1, TMED9, AARS, SDF2L1, EPN1, PFN1, DUFS6, NDUFB9, GLTPD2, PSMB4, GCHFR, SPCS3, ID1, RABAC1, RTKN, COX8A, PSMC5, GLTSCR2, SF3B5, RPS20, NDUFB2, NEDD8, JUNB, MYL6, EEF2, ARL5B, TOMM7, FAM96B, SAA2, RPL11, TRAPPC1, EDF1, Cl lorflO, ATP5D, RPL35A, GDI2, SURF4, RPL31, G0S2, SEC61G, NOLA3, SAA4, VAMP 8, IFITM3, C8G, and RPL37A.
5. The method of any one of the previous claims, wherein the one or more sentinel RNAs are one or both of GNB2L1 and TMBIM6.
6. The method of any one of the previous claims, wherein the amount of degradation comprises one or more selected from the group consisting of: 3 '-5' processive degradation, 5 '-3 ' processive degradation, a ratio of 3 '-5 ' processive degradation to 5 '-3' processive degradation, and a ratio of 5 '-3 ' processive degradation to 3 '-5' processive degradation.
7. The method of claim 6, wherein the 3 '-5 ' processive degradation is determined from 200 nucleotides from the 3 '-end of the one or more sentinel RNAs.
8. The method of claim 7, wherein the 5 '-3 ' processive degradation is determined from 200 nucleotides from the 5 '-end of the one or more sentinel RNAs.
9. The method of any one of the previous claims, wherein the measuring is by RNA sequencing.
10. The method of any of the previous claims, wherein the measuring is by quantitative PCR.
11. The method of claim 10, wherein the quantitative PCR is performed using primers specific for the one or more sentinel RNAs selected from the group consisting of EIF4G2, STOM, C4BPA, GNBl, TMEM66, NAGS, CALM2, ACTGl, AGT, EIF1, SCARB1, C20orO, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT, SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, ALB, CLNS1A, ARF1, NDUFV1, RPL10A, SDC4, LASP1, ECHS1, C19orf63, FGG, TNFRSF1A, EIF4EBP1, CDC42EP1, RPN1, HLA-B, TFR2, SERPINA3, TMEM111, TMSB4X, AC025165.27, AMBP, ACAT1, SEC61A1, BRP44L, CTSB, MARS, PCK1, DHCR24, KNG1, TUBA IB, SLC27A5, UBA1, FLU, RHOA, HNF4A, CTSD, IGFBP4, P4HB, LRRC59, SERPINA10, GC, LRPAP1, RPS5, DNAJC3, C4BPB, VTN, IGFBPl, RPN2, BCL2L1, TUBB6, LY6E, FLOT2, NANS, HPN, SERPINF2, SLC9A3R1, ARMET, IDH2, PSMC4, LAPTM4A, GPI, FURIN, APOB, APOH, XBP1, SERPINA11, GUSB, SEMA4B, HPD, KRT18, TMEM176A, RPS2, LRP1, TGM2, FKBPl l, TRIBI, SOD2, AC087521.10, BRI3, GABARAP, CRP, YWHAE, GAPDH, SERPINC1, ITIH1, HMOX1, CHI3L1, C14orf68, HPX, TPT1, HSP90B1, SERPINF1, H19, HSD17B10, ARHGDIA, GDI1, ACTB, KRT8, CFL1,
AF235103.4, HABP2, PLA2G2A, ATP1A1, PRDX2, RPS3, DAD1, METTL7B, AXUD1, PSMD13, HM13, C19orfl0, MORF4L2, FNDC4, SRPR, MSN,
SERPINB1, RPS4X, SAT1, TAGLN2, PSMB7, LAMP1, SERPINA1, CPS1, HSPE1, PSME1, TMEM205, TUBB2C, C8B, RPS13, MLF2, RPL4, PMPCA, UQCRC1, LTBR, PEBP1, APOL1, ATP6V0E, TYMP, RPS9, FTL, AQP3,
SERPINA7, LRG1, GSTK1, ETFB, GRINA, FGA, C21orf33, TCF25, EIF3K, CSTB, C5, CHAC1, EIF3G, IFITM1, APOA4, VPS28, POLD4, F2, ITGA5, DDX17, HSD17B2, SAT2, FGB, PDIA4, ACADVL, IGFBP2, EIF5A, TMBIM6, OAF, ACY1, PHB2, HLA-E, RHOD, ACOl 1498.7, CIB1, RPL12, GRHPR, RARRES2, TTR, GPX3, CD74, RPL30, HRG, RASD1, C9, SSR2, SHC1, C3, AZGPl, RRBPl, CALR, NDUFA4, GNB2L1, C19orf43, MDH2, GSTOl, LMANl, APCS, PSMD4, SDS, PPIB, AP001453.6, BLOCI SI, RPL35, GADD45B, RPLPl, TMED9, AARS, SDF2L1, EPN1, PFN1, DUFS6, NDUFB9, GLTPD2, PSMB4, GCHFR, SPCS3, ID1, RABAC1, RTKN, COX8A, PSMC5, GLTSCR2, SF3B5, RPS20, NDUFB2, NEDD8, JUNB, MYL6, EEF2, ARL5B, TOMM7, FAM96B, SAA2, RPL11, TRAPPC1, EDF1, Cl lorflO, ATP5D, RPL35A, GDI2, SURF4, RPL31, G0S2, SEC61G, NOLA3, SAA4, VAMP 8, IFITM3, C8G, and RPL37A.
12. The method of claim 11 , wherein the primers specific for the one or more sentinel RNAs are primers specific for one or both of GNB2L1 and TMBIM6.
13. The method of any of the previous claims, wherein the reference standard comprises a known amount of sentinel RNA degradation from a control biological sample.
14. The method of claim 11 , wherein the primers specific for the one or more sentinel RNAs are primers specific for SERPINA3, B2M, ENOl, GNB2L1, TMBIM6, PFN1, MYL6, SAA2, JUNB and TIMP1.
15. The method of any one of the previous claims, wherein the amount of degradation is measured at one or more time points.
16. The method of any one of the previous claims, wherein the indentifying comprises isolating 5 '-capped RNA in the biological sample and determining the level of degradation at one or more time points.
17. A method of identifying one or more sentinel RNAs in a biological sample, the
method comprising:
(a) isolating RNA from one or more abundantly expressed genes in the biological sample;
(b) measuring processive degradation of the isolated RNA at one or more time points; and
(c) classifying the one or more abundantly expressed genes as the one or more sentinel RNAs based on the measuring.
18. The method of claim 17, wherein the isolated RNA is 5 '-capped RNA.
19. The method of any one of claims 17-18, wherein the isolated RNA is mRNA.
20. The method of any one of claims 17-19, wherein the processive degradation is one or more selected from the group consisting of: 3 '-5' processive degradation, 5 '-3' processive degradation, a ratio of 3 '-5' processive degradation to 5 '-3' processive degradation, and a ratio of 5 '-3' processive degradation to 3 '-5' processive degradation.
21. The method of claim 20, wherein the 3 '-5 ' processive degradation is from 200
nucleotides from the 3 '-end of the one or more sentinel RNAs.
22. The method of claim 20, wherein the 5 '-3 ' processive degradation is from 200
nucleotides from the 5 '-end of the one or more sentinel RNAs.
23. The method of any one of claims 17-22, wherein the processive degradation is rapid compared to the processive degradation from a control RNA.
24. The method of any one of claims 17-23, wherein the measuring is by RNA
sequencing.
25. The method of any one of claims 17-24, wherein the measuring is by quantitative PCR.
26. The method of any one of claims 17-25, wherein the sentinel RNA comprises one or more selected from the group consisting of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orf3, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLP0, CRAT, SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, ALB, CLNS1A, ARF1, NDUFV1, RPL10A, SDC4, LASP1, ECHS1, C19orf63, FGG, TNFRSF1A, EIF4EBP1, CDC42EP1, RPN1, HLA-B, TFR2, SERPINA3, TMEMl 11, TMSB4X, AC025165.27, AMBP, ACATl, SEC61A1, BRP44L, CTSB, MARS, PCKl, DHCR24, KNGl, TUBAIB, SLC27A5, UBA1, FLU, RHOA, HNF4A, CTSD, IGFBP4, P4HB, LRRC59, SERPINA10, GC, LRPAP1, RPS5, DNAJC3, C4BPB, VTN, IGFBP1, RPN2, BCL2L1, TUBB6, LY6E, FLOT2, NANS, HPN, SERPINF2, SLC9A3R1, ARMET, IDH2, PSMC4, LAPTM4A, GPI, FURIN, APOB, APOH, XBP1, SERPINA11, GUSB, SEMA4B, HPD, KRT18, TMEM176A, RPS2, LRP1, TGM2, FKBP11, TRIB1, SOD2, AC087521.10, BRI3, GABARAP, CRP, YWHAE, GAPDH, SERPINC1, ITIH1, HMOX1, CHI3L1, C14orf68, HPX, TPT1, HSP90B1, SERPINF1, H19,
HSD17B10, ARHGDIA, GDI1, ACTB, KRT8, CFL1, AF235103.4, HABP2, PLA2G2A, ATP1A1, PRDX2, RPS3, DAD1, METTL7B, AXUD1, PSMD13, HM13, C19orfl0, MORF4L2, FNDC4, SRPR, MSN, SERPINB1, RPS4X, SAT1, TAGLN2, PSMB7, LAMP1, SERPINA1, CPS1, HSPE1, PSME1, TMEM205, TUBB2C, C8B, RPS13, MLF2, RPL4, PMPCA, UQCRCl, LTBR, PEBPl, APOLl, ATP6V0E, TYMP, RPS9, FTL, AQP3, SERPINA7, LRG1, GSTK1, ETFB, GRINA, FGA, C21or03, TCF25, EIF3K, CSTB, C5, CHAC1, EIF3G, IFITM1, APOA4, VPS28, POLD4, F2, ITGA5, DDX17, HSD17B2, SAT2, FGB, PDIA4, ACADVL, IGFBP2, EIF5A, TMBIM6, OAF, ACY1, PHB2, HLA-E, RHOD, ACOl 1498.7, CIB1, RPL12, GRHPR, RARRES2, TTR, GPX3, CD74, RPL30, HRG, RASD1, C9, SSR2, SHC1, C3, AZGP1, RRBP1, CALR, NDUFA4,
GNB2L1, C19orf43, MDH2, GSTOl, LMAN1, APCS, PSMD4, SDS, PPIB, AP001453.6, BLOC1S1, RPL35, GADD45B, RPLP1, TMED9, AARS, SDF2L1, EPN1, PFN1, DUFS6, NDUFB9, GLTPD2, PSMB4, GCHFR, SPCS3, ID1, RABAC1, RTKN, COX8A, PSMC5, GLTSCR2, SF3B5, RPS20, NDUFB2, NEDD8, JUNB, MYL6, EEF2, ARL5B, TOMM7, FAM96B, SAA2, RPL11, TRAPPC1, EDF1, Cl lorflO, ATP5D, RPL35A, GDI2, SURF4, RPL31, G0S2, SEC61G, NOLA3, SAA4, VAMP 8, IFITM3, C8G, and RPL37A.
The method of claim 25, wherein the quantitative PCR is performed using primers specific for the one or more sentinel RNAs selected from the group consisting of EIF4G2, STOM, C4BPA, GNBl, TMEM66, NAGS, CALM2, ACTGl, AGT, EIF1, SCARBl, C20orf3, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLPO, CRAT, SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, ALB, CLNS1A, ARF1, NDUFV1, RPL10A, SDC4, LASP1, ECHS1, C19orf63, FGG, TNFRSF1A, EIF4EBP1, CDC42EP1, RPNl, HLA-B, TFR2, SERPINA3, TMEMl l l, TMSB4X, AC025165.27, AMBP, ACAT1, SEC61A1, BRP44L, CTSB, MARS, PCK1, DHCR24, KNG1, TUBA IB, SLC27A5, UBA1, FLU, RHOA, HNF4A, CTSD, IGFBP4, P4HB, LRRC59, SERPINA10, GC, LRPAP1, RPS5, DNAJC3, C4BPB, VTN, IGFBPl, RPN2, BCL2L1, TUBB6, LY6E, FLOT2, NANS, HPN, SERPINF2, SLC9A3R1, ARMET, IDH2, PSMC4, LAPTM4A, GPI, FURIN, APOB, APOH, XBP1, SERPINA11, GUSB, SEMA4B, HPD, KRT18, TMEM176A, RPS2, LRP1, TGM2, FKBPl l, TRIBI, SOD2, AC087521.10, BRI3, GABARAP, CRP, YWHAE, GAPDH, SERPINCl, ITIHI, HMOXl, CHI3L1, C14orf68, HPX, TPTl, HSP90B1, SERPINF1, H19, HSD17B10, ARHGDIA, GDI1, ACTB, KRT8, CFL1,
AF235103.4, HABP2, PLA2G2A, ATP1A1, PRDX2, RPS3, DAD1, METTL7B, AXUD1, PSMD13, HM13, C19orfl0, MORF4L2, FNDC4, SRPR, MSN,
SERPINB1, RPS4X, SAT1, TAGLN2, PSMB7, LAMP1, SERPINA1, CPS1, HSPE1, PSME1, TMEM205, TUBB2C, C8B, RPS13, MLF2, RPL4, PMPCA, UQCRC1, LTBR, PEBP1, APOL1, ATP6V0E, TYMP, RPS9, FTL, AQP3,
SERPINA7, LRG1, GSTK1, ETFB, GRINA, FGA, C21orf33, TCF25, EIF3K, CSTB, C5, CHAC1, EIF3G, IFITM1, APOA4, VPS28, POLD4, F2, ITGA5, DDX17, HSD17B2, SAT2, FGB, PDIA4, ACADVL, IGFBP2, EIF5A, TMBIM6, OAF, ACY1, PHB2, HLA-E, RHOD, ACOl 1498.7, CIB1, RPL12, GRHPR, RARRES2, TTR, GPX3, CD74, RPL30, HRG, RASD1, C9, SSR2, SHC1, C3, AZGPl, RRBPl, CALR, NDUFA4, GNB2L1, C19orf43, MDH2, GSTOl, LMANl, APCS, PSMD4, SDS, PPIB, AP001453.6, BLOC1 S1, RPL35, GADD45B, RPLP1, TMED9, AARS, SDF2L1, EPN1, PFN1, DUFS6, NDUFB9, GLTPD2, PSMB4, GCHFR, SPCS3, ID1, RABAC1, RTKN, COX8A, PSMC5, GLTSCR2, SF3B5, RPS20, NDUFB2, NEDD8, JUNB, MYL6, EEF2, ARL5B, TOMM7, FAM96B, SAA2, RPL11, TRAPPC1, EDF1, Cl lorflO, ATP5D, RPL35A, GDI2, SURF4, RPL31, G0S2, SEC61G, NOLA3, SAA4, VAMP 8, IFITM3, C8G, and RPL37A.
The method of claim 27, wherein the primers specific for the one or more sentinel RNAs are primers specific for one or both of GNB2L1 and TMBIM6.
The method of claim 17, wherein the biological sample is selected from the group consisting of blood, plasma, serum, lymph, mucus, sputum, tears, urine, stool, saliva, tissue, hair, animal cells, and plant cells.
30. A kit for testing the integrity of a biological sample, the kit comprising:
(a) one or more primers specific for one or more sentinel RNAs;
(b) optionally, reagents for qPCR or RNA sequencing; and
(c) a degradation reference standard.
31. The kit of claim 30, wherein the one or more primers are specific for the one or more sentinel RNAs selected from the group consisting of EIF4G2, STOM, C4BPA, GNB1, TMEM66, NAGS, CALM2, ACTG1, AGT, EIF1, SCARB1, C20orf3, CNDP2, CD81, ATP5B, AGXT, EIF4A1, HSP90AB1, AHSG, SARS, CDKN1A, SEPHS2, PSAP, RPL5, TGFBI, CPB2, CCND1, AP2M1, ERGIC3, IBTK, YIF1A, ENOl, UGT2B4, GABARAPL1, KDELR2, RHBDD2, CD63, F9, TRAM1, DHRS3, ZCCHC24, SNRPB, LPCAT3, B2M, ITIH3, RPLPO, CRAT, SERPINA5, ATP5C1, RPL13A, LBP, GSDMD, ALB, CLNS1A, ARF1, NDUFV1, RPL10A, SDC4, LASP1, ECHS1, C19orf63, FGG, TNFRSF1A, EIF4EBP1, CDC42EP1, RPN1, HLA-B, TFR2, SERPINA3, TMEM111, TMSB4X, AC025165.27, AMBP, ACAT1, SEC61A1, BRP44L, CTSB, MARS, PCK1, DHCR24, KNG1, TUBA IB, SLC27A5, UBA1, FLU, RHOA, HNF4A, CTSD, IGFBP4, P4HB, LRRC59, SERPINA10, GC, LRPAP1, RPS5, DNAJC3, C4BPB, VTN, IGFBP1, RPN2, BCL2L1, TUBB6, LY6E, FLOT2, NANS, HPN, SERPINF2, SLC9A3R1, ARMET, IDH2, PSMC4, LAPTM4A, GPI, FURIN, APOB, APOH, XBP1, SERPINA11, GUSB, SEMA4B, HPD, KRT18, TMEM176A, RPS2, LRP1, TGM2, FKBP11, TRIB1, SOD2, AC087521.10, BRI3, GABARAP, CRP, YWHAE, GAPDH, SERPINC1, ITIH1, HMOX1, CHI3L1, C14orf68, HPX, TPT1, HSP90B1,
SERPINF1, H19, HSD17B10, ARHGDIA, GDI1, ACTB, KRT8, CFL1,
AF235103.4, HABP2, PLA2G2A, ATP1A1, PRDX2, RPS3, DAD1, METTL7B, AXUD1, PSMD13, HM13, C19orfl0, MORF4L2, FNDC4, SRPR, MSN,
SERPINB1, RPS4X, SAT1, TAGLN2, PSMB7, LAMP1, SERPINA1, CPS1, HSPE1, PSME1, TMEM205, TUBB2C, C8B, RPS13, MLF2, RPL4, PMPCA, UQCRC1, LTBR, PEBP1, APOL1, ATP6V0E, TYMP, RPS9, FTL, AQP3, SERPINA7, LRG1, GSTK1, ETFB, GRINA, FGA, C21orf33, TCF25, EIF3K, CSTB, C5, CHAC1, EIF3G, IFITM1, APOA4, VPS28, POLD4, F2, ITGA5, DDX17, HSD17B2, SAT2, FGB, PDIA4, ACADVL, IGFBP2, EIF5A, TMBIM6, OAF, ACY1, PHB2, HLA-E, RHOD, ACOl 1498.7, CIB1, RPL12, GRHPR, RARRES2, TTR, GPX3, CD74, RPL30, HRG, RASD1, C9, SSR2, SHC1, C3, AZGPl, RRBPl, CALR, NDUFA4, GNB2L1, C19orf43, MDH2, GSTOl, LMANl, APCS, PSMD4, SDS, PPIB, AP001453.6, BLOC1 S1, RPL35, GADD45B, RPLP1, TMED9, AARS, SDF2L1, EPN1, PFN1, DUFS6, NDUFB9, GLTPD2, PSMB4, GCHFR, SPCS3, ID1, RABAC1, RTKN, COX8A, PSMC5, GLTSCR2, SF3B5, RPS20, NDUFB2, NEDD8, JUNB, MYL6, EEF2, ARL5B, TOMM7, FAM96B, SAA2, RPL11, TRAPPC1, EDF1, Cl lorflO, ATP5D, RPL35A, GDI2, SURF4, RPL31, G0S2, SEC61G, NOLA3, SAA4, VAMP 8, IFITM3, C8G, and RPL37A.
The kit of claim 30, wherein the primers specific for the one or more sentinel RNAs are primers specific for one or both of GNB2L1 and TMBIM6.
The kit of claim 30, wherein the primers specific for the one or more sentinel RNAs are primers specific for SERPINA3, B2M, ENOl, GNB2L1, TMBIM6, PFN1, MYL6, SAA2, JUNB and TIMP1.
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