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WO2025034736A1 - Préparation d'échantillon pour acides nucléiques - Google Patents

Préparation d'échantillon pour acides nucléiques Download PDF

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Publication number
WO2025034736A1
WO2025034736A1 PCT/US2024/041083 US2024041083W WO2025034736A1 WO 2025034736 A1 WO2025034736 A1 WO 2025034736A1 US 2024041083 W US2024041083 W US 2024041083W WO 2025034736 A1 WO2025034736 A1 WO 2025034736A1
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Prior art keywords
tergitol
acid
lysis buffer
triton
brij
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Erik QUANDT
Laura MIER
Cynthia BOARDMAN
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Life Technologies Corp
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Life Technologies Corp
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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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/10—Nucleotidyl transfering
    • C12Q2521/107—RNA dependent DNA polymerase,(i.e. reverse transcriptase)
    • 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
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/301—Endonuclease
    • 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
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/327—RNAse, e.g. RNAseH
    • 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
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/125—Specific component of sample, medium or buffer
    • 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
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/137—Concentration of a component of medium

Definitions

  • the present teachings generally relate to compositions, processes, methods, and kits for preparation of samples containing genetic material for downstream analysis, such as detection and/or quantitation.
  • PCR real-time polymerase chain reaction
  • RT-qPCR real-time quantitative reverse transcription-PCR
  • Many procedures for nucleic acid preparation contain components that are inhibitory for optimum reverse transcriptase function or for optimum DNA polymerase function, and/or contain components that are not REACH compliant.
  • the present teachings provide improved compositions, methods, and kits for preparation of samples for downstream analysis, including detection and/or quantitation of nucleic acids.
  • teachings herein include a method for preparing a sample containing nucleic acids, e.g., for downstream analysis.
  • the method includes contacting the sample containing nucleic acids with a lysis buffer to produce a lysis mixture, and incubating the lysis mixture and for a period of time.
  • the lysis mixture is incubated at a temperature (lysis temperature), e.g., from about 5 °C to about 40 °C, from about 15 °C to about 30 °C, from about 16 °C to about 28 °C or from about 19 °C to about 25 °C as further described infra for a period of time (lysis time), e.g., between at least 1 minute to one hour or longer (for example, up to 24 hours).
  • lysis temperature e.g., from about 5 °C to about 40 °C, from about 15 °C to about 30 °C, from about 16 °C to about 28 °C or from about 19 °C to about 25 °C as further described infra
  • lysis time e.g., between at least 1 minute to one hour or longer (for example, up to 24 hours).
  • the lysis buffer can include one or more anionic oligomers having RNase inhibitory activity, and one or more surfactant.
  • the anionic oligomer with RNase activity can be, for example, Poly (vinyl sulfonic acid) (PVSA), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo- 2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly(4-styrenesulfonic acid), or Dextran sulfate, or any combination thereof.
  • PVSA Poly (vinyl sulfonic acid)
  • PVSA Poly(vinylphosphonic acid)
  • Sulfated nitrochitin Sulfated nitrochitosan
  • Sulfated polyvinyl alcohol Heparin
  • Fucoidan Poly(2-acrylamindo- 2-methyl-l -propanesulfonic acid)
  • Polyanetholesulfonic acid Poly(4-sty
  • the one or more surfactants substantially lack fluorescence between 300 nm and 750 nm at a lysis-effective concentration, e.g., between 0.05% to 4.0% (v/v) of the lysis buffer. More preferably, the one or more surfactants are REACH compliant.
  • the one or more surfactants can be selected from anion surfactants, cationic surfactants, zwitterionic surfactants, and non-ionic surfactants.
  • the one or more surfactants can be selected from: Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, ECOSURFTM EH-9, EcosurfTM SA-4, EcosurfTM SA-9, EcosurfTM EH-6, EcosurfTM EH-3, Brij® 35, Brij® 58, Brij® L23, Brij® S10 sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, and ammonium laureth sulfate, or any combination thereof.
  • the lysis mixture is substantially free of a chelator. In other embodiments, a chelator is present in the lysis buffer.
  • the lysis mixtures described herein are compatible with in situ polymerase and reverse transcriptase reactions.
  • the lysis buffer can include a DNase, such as a heat-labile double strand specific DNase (HL-dsDNase).
  • DNase e.g., a HL- dsDNase
  • HL-dsDNase heat-labile double strand specific DNase
  • the lysis buffer can include a RNase inhibitor protein.
  • RNase inhibitor protein is added to the lysis mixture.
  • the concentration of the RNase inhibitor protein in the lysis buffer would be 0.1 U/uL and 4 U/uL.
  • a unit is defined as the amount of Ribonuclease Inhibitor required to inhibit the activity of 5ng of ribonuclease A by 50%.
  • Common RNase inhibitor protein sources include porcine liver, bovine pancreas, dormouse, murine, human placenta, and rat lung. These can either by native (purified from host organism) or recombinant (expressed and purified from a different organism).
  • the lysis buffer further can include a salt such as an alkaline earth metal salt, including but not limited to magnesium chloride, calcium chloride, or a combination thereof.
  • Preferable lysis buffers include an anionic oligomer having RNase inhibitory activity, one or more surfactants, and one or more salts.
  • a DNase is added to a lysis mixture that comprises the lysis buffer.
  • the lysis mixture can be further combined with reagents for reverse transcription (RT) to form an RT product and, in some embodiments, the RT product is contacted with reagents for amplification, including but not limited to quantitative polymerase chain reaction (qPCR) amplification.
  • RT reverse transcription
  • qPCR quantitative polymerase chain reaction
  • the reagents for reverse transcription and amplification can include, for example, a buffer, enzymes (e.g, reverse transcriptase, polymerase, etc.), nucleotides, and the like.
  • the cell lysate does not need to be treated or further processed prior to using the cell lysate in in situ reactions, such as reverse transcription or RT-qPCR, but used directly in such downstream processes.
  • nucleic acids for in situ analysis from a sample that contains nucleic acids (e.g., a biological or environmental sample or the like). Accordingly, provided herein are methods for preparing RNA from a sample containing nucleic acids. The method can include contacting the sample containing nucleic acids with a lysis buffer to produce a lysis mixture, and incubating the lysis mixture at a lysis temperature of about 16 °C to about 40 °C for a lysis time that is at least one minute to produce a cell lysate.
  • the lysis buffer can include an anionic oligomer having RNase inhibitory activity, e.g., Poly(vinyl sulfonic acid) (PVSA), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo- 2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly (4- styrenesulfonic acid), k- Carrageenan, i-Carrageenan, X-Carragccnan.
  • PVSA Poly(vinyl sulfonic acid)
  • PVPA Poly(vinylphosphonic acid)
  • Sulfated nitrochitin Sulfated nitrochitosan
  • Sulfated polyvinyl alcohol Heparin, Fucoidan
  • the lysis buffer can also include one or more surfactants such as Tergitol 15-S-9, Tergitol 15-S-12, CHAPS (3-((3-cholaniidopropyl) dimethylammonio)- 1 -propanesul foriate, CHAPSO (3-([3-
  • Zwittergent® 3- 14 Cholamidopropyl]dimetby1ammonio)-2-bydroxy- 1 -propanesulfonate
  • Zwittergent® 3- 14 n- Tetradecyl-N,N-dimethyl-3-ammonio-l-propanesulfonate
  • Zwittergent® 3-12 zz-Dodecyl- N,N-dimethyl-3-ammonio-l -propanesulfonate
  • Zwittergent® 3-16 n-Hexadecyl-N,N- dimcthyl-3-ammonio-l-propancsulfonatc
  • Zwittergent® 3-08 n-Octyl-N,N-dimcthyl-3- ammonio-1 -propanesulfonate
  • Zwittergent® 3-10 (7z-Decyl-N,N-dimethyl-3-ammonio-l- propanesulfonate), sodium dodecy
  • the lysis buffer further can include one or more salts such as an alkaline earth metal salt, including but not limited to magnesium chloride, calcium chloride, or a combination thereof.
  • the lysis buffer is substantially free of a chelator.
  • the lysis mixture can be contacted with a DNase such as a double- stranded DNase.
  • the DNase can be added to the lysis mixture.
  • the DNase is included in the lysis buffer.
  • the DNase can be a HL-dsDNase.
  • the lysis mixture can be contacted with a RNase inhibitor protein.
  • the RNase inhibitor protein can be added to the lysis mixture.
  • the RNase inhibitor protein is included in the lysis buffer.
  • the lysis buffer and/or lysis mixture can be contacted with a polypeptide possessing protease activity or mixtures of polypeptides with protease activity to facilitate cell dissociation and lysis.
  • the proteases can be, for example, Trypsin, Pepsin, Proteinase K, Papain, Dispase I, Dispase II, Collagenase I, Collagenase II, Collagenase III, Collagenase IV, Collagenase V, Collagenase VI, Collagenase VII, Collagenase VIII, Collagenase XI, Accutase.
  • the lysis buffer can include a protease or mixture of proteases.
  • a protease or mixture of proteases is added to the lysis mixture.
  • the resulting cell lysate can be compatible with in situ polymerase and reverse transcriptase reactions, e.g., without further processing or extracting the cell lysate.
  • a method for preparing total nucleic acids from a sample can include contacting the sample containing nucleic acids with a lysis buffer to produce a lysis mixture, and incubating the lysis mixture at about 16 °C to about 40 °C for a period of time to produce a cell lysate.
  • the lysis buffer comprises an anionic oligomer having RNase inhibitory activity selected from the group consisting of Poly(vinyl sulfonic acid) (PVSA), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2- acrylamindo-2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly(4- styrenesulfonic acid), k-Carrageenan, i-Carrageenan, A-Carrageenan, Poly(4-styrenesulfonic acid-co -maleic acid), and Dextran sulfate, and a surfactant at a concentration in the lysis buffer of 0.05% to 0.3% selected from the group consisting of Tergitol 15-S-9, Tergitol 15-S-12, sodium dodecyl sulf
  • the method can include contacting the sample containing RNA with a lysis buffer to produce a lysis mixture; and incubating the lysis mixture at an incubation temperature and for a time to produce a cell lysate, wherein the lysis buffer comprises, an anionic oligomer having RNase inhibiting properties; and a surfactant; and wherein the cell lysate is compatible with in situ polymerase or reverse transcription reactions.
  • the method further comprises contacting the lysis mixture with a double- stranded DNase.
  • the double-stranded DNase comprises a heat-labile double-strand specific DNase (HL-dsDNase).
  • the method further comprises contacting the lysis mixture with a RNase inhibitor protein.
  • the method further comprises contacting the cell lysate with reagents for reverse transcription to produce an RT product. In some embodiments, the method further comprises contacting the RT product with reagents for qPCR amplification. [0018] In some embodiments, the method further comprises wherein the contacting is carried out at about 5 °C to about 40 °C. In some embodiments, the method further comprises wherein the contacting is carried out at ambient temperature.
  • the method further comprises a sample, wherein the sample comprises a cell or cell culture.
  • the cell culture has been cultured on extracellular matrix.
  • the cell culture comprises primary cells.
  • the primary cells comprise primary hepatocytes.
  • the cells are selected from the group consisting of Kupffer cells, PBMCs, THP-1 cells, HL60 cells or any combination thereof.
  • the method further comprises a sample, wherein the sample is a tissue sample.
  • RNA from a sample containing nucleic acids comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises an anionic oligomer, wherein the anionic oligomer is selected from the group consisting of Poly(vinylphosphonic acid), Polyanetholesulfonic acid, Poly(4-styrenesulfonic acid-co-maleic acid), Poly(vinyl sulfonic acid), Poly(4- styrenesulfonic acid), or any combination thereof.
  • the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer further comprises a surfactant, wherein the surfactant is selected from the group consisting of Tergitol 15-S-9, Tergitol 15-S-12, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, Ecosurf IM SA-9, Ecosurf 1 "' EH- 6, EcosurfTM EH-3, EcosurfTM SA-7, TRITON X-114TM, TRITON X-100TM, or any combination thereof.
  • the surfactant is selected from the group consisting of Tergitol 15-S-9, Tergitol 15-S-12, sodium dodecyl sulfate, sodium laureth
  • the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer further comprises a surfactant, wherein the surfactant is a cationic surfactant, an anionic surfactant, a non-ionic surfactant, a zwitterionic surfactant, or any combination thereof.
  • a method for preparing RNA from a sample containing nucleic acids is provided, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a surfactant, wherein the surfactant is a cationic surfactant.
  • a method for preparing RNA from a sample containing nucleic acids comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a surfactant, wherein the surfactant is a non-ionic surfactant.
  • a method for preparing RNA from a sample containing nucleic acids is provided, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a surfactant, wherein the surfactant is a zwitterionic surfactant.
  • a method for preparing RNA from a sample containing nucleic acids comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a surfactant, wherein the surfactant is an anionic surfactant.
  • a method for preparing RNA from a sample containing nucleic acids comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises an anionic surfactant, wherein the anionic surfactant is selected from the group consisting of sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, ammonium laureth sulfate, or any combination thereof.
  • the concentration of anionic surfactant in the lysis buffer is 0.05% to 0.3%.
  • a method for preparing RNA from a sample containing nucleic acids comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a cationic surfactant, wherein the cationic surfactant is selected from the group consisting of cetyl trimethylammonium bromide (CTAB), cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2- hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethylcellulose ethoxylate, polyquatemium-10, and hexadecyl-trimethylammoniumchloride (HTAC), or any combination thereof.
  • CTAB cetyl trimethylammonium bromide
  • CTAC cetyl trimethylammonium chloride
  • CPC cetylpyridinium chloride
  • HTAC hexadecyl-trimethylammoniumchloride
  • a method for preparing RNA from a sample containing nucleic acids comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a nonionic surfactant, wherein the nonionic surfactant is selected from the group consisting of Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S- 12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP- 1 1 , Tergitol NP-13, Tergitol NP-50, Tergitol NP-30, EcosurfTM SA-9, EcosurfTM EH-6, EcosurfTM EH-3, EcosurfTM SA-7, TRITON X-114TM, TRITON X-100TM, or any combination thereof.
  • the concentration of nonionic surfactant in the lysis buffer is 0.05% to 0.3%.
  • a method for preparing RNA from a sample containing nucleic acids comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a zwitterionic surfactant, wherein the zwitterionic surfactant is selected from the group consisting of CHAPS (3-((3-cholamidopropyl) dimethylammonio)- 1 -propanesulfonate, CH APSO (3-([3-
  • the method can include preparing RNA according to the methods in any of the embodiments described above and using the RNA prepared in a reverse transcription reaction, wherein the RNA prepared is not treated with a stop solution prior to using the prepared RNA in a reverse transcription reaction.
  • RNA from a sample comprising cells comprising contacting the sample with a lysis buffer to produce a lysis mixture; and incubating the lysis mixture at about 16 °C to about 28 °C for a period of time to produce a cell lysate comprising RNA
  • the lysis buffer comprises; (i) an anionic oligomer having RNase inhibiting properties selected from the group consisting of: Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo-2-methyl- 1 -propanesulfonic acid), Polyanetholesulfonic acid, Poly(vinyl sulfonic acid), Poly(4-styrenesulfonic acid), and Dextran sulfate; (ii) a surfactant selected from the group consisting of sodium dodecy
  • the method further comprises contacting the lysis mixture with a heat-labile, double- stranded DNase.
  • the method further comprises contacting the lysis mixture with a RNase inhibitor.
  • the RNase inhibitor is an RNase inhibitor protein, a non-proteinaceous RNase inhibitor, or combination thereof.
  • the non- proteinaceous RNase inhibitor is selected from ADP, a vanadyl complex, or combination thereof.
  • kits for preparation nucleic acids from a sample containing nucleic acids can include a lysis buffer comprising an anionic oligomer having RNase inhibitory activity (e.g, Poly(vinyl sulfonic acid) (PVSA), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo-2-methyl- 1 -propanesulfonic acid), Polyanetholesulfonic acid, Poly(4- styrenesulfonic acid), k-Carrageenan, i-Carrageenan, X-Carrageenan, Poly(4-styrenesulfonic acid-co -maleic acid), Dextran sulfate, or any combination thereof), and a surfactant at a concentration in the lysis buffer of 0.05% to 0.3% (v/v)
  • RNase inhibitory activity e.g
  • kits for preparation nucleic acids from a sample containing nucleic acids can include a lysis buffer comprising an anionic oligomer having RNase inhibitory activity (e.g, Poly(vinyl sulfonic acid) (PVSA), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo-2-methyl- 1 -propanesulfonic acid), Polyanetholesulfonic acid, Poly(4- styrenesulfonic acid), Poly(4-styrenesulfonic acid-co-maleic acid), or any combination thereof), and a surfactant at a concentration in the lysis buffer of 0.05% to 0.3% (v/v) (e.g., Tergitol 15-S-9, Tergitol 15-S-9, Tergitol 15-S-9, Tergitol 15-S-9, Tergit
  • kits for preparation nucleic acids from a sample containing nucleic acids can include a lysis buffer comprising an anionic oligomer having RNase inhibitory activity (e.g, Poly(vinyl sulfonic acid) (PVSA), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo-2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly(4- styrenesulfonic acid), Poly(4-styrenesulfonic acid-co-maleic acid, or any combination thereof), and a surfactant at a concentration in the lysis buffer of 0.05% to 0.3% (v/v) (e.g., sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sul
  • RNase inhibitory activity e.g
  • kits can include one or more reagents for reverse transcription, such as reverse transcriptase, a reverse primer, dNTPs or a reverse transcriptase buffer.
  • reagents for reverse transcription such as reverse transcriptase, a reverse primer, dNTPs or a reverse transcriptase buffer.
  • kits can include one or more reagents for amplification, e.g., PCR, qPCR, rolling circle amplification, isothermal amplification or the like.
  • the kits can include one or more enzymes for amplification, dNTPs, probes, amplification primers, and the like.
  • processes and compositions are compatible with downstream nucleic acid detection methods using methods such as reverse transcription, polymerase chain reaction, qPCR, qRT-PCR, sequencing, message amplification, preamplification using a PREAMP ' 1 kit, detection using a miRNA TAQMAN® probe, linear amplification for array analysis, and others that use CYANINETM 3 or CYANINETM 5 in array analysis, for example.
  • processes and compositions are compatible with downstream detection of miRNA.
  • Sample preparation methods provided herein are useful for preparing nucleic acids for downstream methods wherein RNA or DNA is analyzed, detected or quantitated.
  • compositions and methods described herein surprisingly provide fast, efficient, and ambient temperature production of a lysate that is RT- and PCR-ready due, in part, to provision of conditions under which there is no need of a stop solution.
  • FIG. 1 Provides data demonstrating that a variety of non-ionic surfactants are effective at lysing human cell cultures.
  • HepG2 cells were lysed with buffers containing the non-ionic surfactant indicated or PBS as a negative control.
  • PBS buffers containing the non-ionic surfactant indicated or PBS as a negative control.
  • cellular RNA was extracted and purified using a traditional column-based RNA purification protocol. RNA preparations were subjected to gene expression analysis by RT-qPCR using TAQMAN® probes targeting the IMPA2 gene (5’ FAM-labeled probe), and ROCK2 gene (5’ VIC-labeled probe).
  • FIG. 2 Provides data demonstrating that the addition of PVSA to the cell lysis buffer helps preserve RNA from being degraded over a 20 hr time course.
  • HeLa cells were lysed with lysis buffers containing 75 ug/mL PVSA (white bars) or no PVSA (black bars) and incubated for either 0 hrs, 2 hrs, 5 hrs, or 20 hrs at room temperature.
  • RNA preparations were subjected to gene expression analysis by RT-qPCR using a 5’ FAM-labeled TAQMAN® Gene Expression Assay targeting the PPIA gene. Samples containing PVS A exhibit lower Ct values at each timepoint as compared to samples without PVS A.
  • FIG. 3 Provides data demonstrating that PVS A enhances the digestion of gDNA by HL-dsDNase in cell lysates.
  • PVSA was supplemented to the cell lysis buffer at the concentrations indicated and gDNA content was analyzed using qPCR with a 5’ FAM-labeled TAQMAN® Gene Expression Assay targeting the PPIA gene.
  • FIG. 4 Provides data showing results from sample processing of HeLa cells (10-10 5 cells per lysis reaction) and analysis using a TAQMAN® Gene Expression Assay for CDK4 (black circles) or ACTB (white squares).
  • FIG. 5 Provides data demonstrating that the addition of Collagenase IV to the cell lysis buffer helps lyse Primary Hepatocyte cells grown on a Collagen-coated surface and overlayed with Matrigel extracellular matrix (Corning). Samples containing Collagenase IV in the lysis exhibit lower Ct values as compared to samples without Collagenase IV.
  • FIG. 6 Provides data demonstrating that lysis buffers containing the anionic surfactants (AS-1 and AS-2) are effective at lysing human Primary Hepatocyte and the resulting lysates can be added directly to RT-qPCR reactions producing results comparable to purified RNA.
  • AS-1 and AS-2 anionic surfactants
  • FIG. 7 Provides data demonstrating that Ct values obtained using lysates as prepared using processes provided herein were found to be essentially equivalent to Ct values obtained with purified RNA.
  • ranges are meant to include the starting value and the ending value and a value or value range there between unless otherwise specifically stated.
  • “from 0.2 to 0.5” means 0.2, 0.3, 0.4, 0.5; ranges there between such as 0.2-0.3, 0.3 - 0.4, 0.2 - 0.4; increments there between such as 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between such as 0.26 - 0.39; and the like.
  • A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth.
  • the skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
  • sample refers to an in vitro cell, cell culture, virus, bodily sample, or tissue sample that contains genetic material.
  • the genetic material of the sample comprises RNA.
  • the genetic material of the sample is DNA, or both RNA and DNA.
  • a tissue sample includes a cell isolated from a subject.
  • a subject includes any organism from which a sample can be isolated. Non-limiting examples of organisms include prokaryotes, eukaryotes or archaebacteria, including bacteria, fungi, animals, plants, or protists.
  • the animal can be a mammal or a non-mammal.
  • the mammal can be, for example, a rabbit, dog, pig, cow, horse, human, or a rodent such as a mouse or rat.
  • the tissue sample is a human tissue sample.
  • the tissue sample can be, for example, a blood sample.
  • the blood sample can be whole blood or a blood product (e.g., red blood cells, white blood cells, platelets, plasma, serum).
  • the sample in other non-limiting embodiments, can be saliva, a cheek, throat, or nasal swab, a fine needle aspirate, a tissue print, cerebral spinal fluid, mucus, lymph, feces, urine, skin, spinal fluid, peritoneal fluid, lymphatic fluid, aqueous or vitreous humor, synovial fluid, tears, semen, seminal fluid, vaginal fluids, pulmonary effusion, serosal fluid, organs, bronchio-alveolar lavage, tumors, and constituents and components of in vitro cell cultures.
  • Sample types can be cell lines such as primary cells, primary hepatocytes (plateable, metabolism-grade, transporter-grade, induction-grade), Kupffer cells, PBMCs, THP-1 cells, HL60 cells, or any combination thereof.
  • the tissue sample is a solid tissue sample.
  • the sample comprises a virus, bacteria, or fungus.
  • the sample can be an ex vivo tissue or sample.
  • the sample can be a fixed sample, including as set forth by U.S. Published Patent Application No. 2003/0170617 filed January 28, 2003.
  • sample types useful in the embodiments provided herein include, for example, saliva, nasal swab, nasopharyngeal swab, buccal swab, rectal swab, vaginal swab, sputum, urine, stool, blood, tissue, and semen, environmental samples (e.g., wastewater, sewage, or the like), or any combination thereof (e.g., a nasopharyngeal swab and saliva), agricultural sample, or derived from animals.
  • the compositions and methods provided herein are useful for preparation of nucleic acids from samples containing, e.g., from one cell up to about 5 x 10 6 cells per sample or any range therebetween. For example, a patient needle biopsy often consists of thousands of cells. A biopsy could be prepared using methods herein, PCR amplified and analyzed by measuring the expression of certain genes, for example.
  • the samples can be pre-treated prior to the processes described herein.
  • cells can be separated from serum components prior to methods provided herein.
  • the sample is washed with a solution comprising, for example, but not limited to, phosphate-buffered saline (PBS), physiological saline, serum-free media or suitable solution with appropriate tonicity.
  • PBS phosphate-buffered saline
  • Samples can also be concentrated, e.g., by centrifugation, washed, etc. prior to processing according to the methods provided herein.
  • the samples can be provided in a minimal volume, e.g., less than 25 pl, preferably less than 10 pl (e.g., 5 pl or less).
  • the volume of lysis buffer used to contact the sample is more than 5-fold, e.g., 10-fold the volume of the sample.
  • RNA or DNA need not be isolated from the cell lysate prior to mixing at least a portion of the cell lysate with a composition comprising reverse transcriptase or another relevant enzyme.
  • a surrogate thereof means a detectable product that represents the RNA or DNA present in the sample, such as an amplified product of the RNA or DNA.
  • a “lysis mixture,” as used herein, refers to the combination of a sample with a lysis buffer, wherein the lysis buffer includes components for lysing cells, viruses, or the like present in the sample.
  • the lysis buffer and lysis mixture lack components that can interfere with downstream processing of nucleic acids, e.g., reverse transcription and/or amplification reactions.
  • the lysis buffer and lysis mixture also lack components that could interfere with methods of detecting nucleic acids using emission detection at wavelengths of 300 nm to 750 nm.
  • the cell lysates e.g., produced by incubating the lysis mixtures as described herein preferably do not require further processing (e.g., inactivation of enzymes), prior to use in downstream analyses such as reverse transcription and/or amplification reactions.
  • the compositions and methods provided herein arc faster and simpler when compared to traditional sample preparation processes (c.g., lysis with harsh chemicals that must be removed prior to use of the nucleic acids in downstream reactions), making the methods provided herein suitable for automation, and high throughput applications.
  • the lysis mixtures described herein are incubated for a period of time, which can range from 1 minute to several hours, depending upon the incubation temperature.
  • the lysis mixtures can be incubated for a period of time between 1 minute and 2 hours, at about 16 °C to 28 °C.
  • lysis mixtures can be incubated at 16 °C to 28 °C for 2 minutes to about 60 minutes, about 2 minutes to about 20 minutes, about 3 minutes to about 15 minutes, about 4 minutes to about 10 minutes or about 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, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes 34 minutes 35 minutes 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes 42 minutes 43 minutes, 44 minutes 45 minutes, 46 minutes 47 minutes, 48 minutes 49 minutes, 50 minutes, 51 minutes, 52 minutes 53 minutes 54 minutes 55 minutes 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, or longer, or any time in between.
  • lysis mixtures can be held on ice, or incubated at 4 °C for 15 minutes to 12 hours or longer, e.g., 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer, or any time in between.
  • Temperature The methods provided herein include incubation of the lysis mixture at a temperature, which is preferably between about 15 °C to 40 °C, or about 16 °C to 28 °C or about 19 °C to 26 °C, or about 19 °C to 25 °C, or about 22 °C to 25 °C, or at ambient temperature, or about 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C,
  • the lysis mixture remains at substantially the same temperature during the incubation time.
  • “Substantially the same temperature” generally refers to an isothermal process of holding the temperature relatively constant during the incubation time, for certain embodiments described herein, means ambient temperature which temperature may change during the day or from lab to lab. An isothermal process is particularly amenable for high throughput analyses.
  • the incubation temperature is such that the HL-dsDNase is not inactivated (e.g., below 50°C).
  • Lysis buffers provided herein include an anionic oligomer having RNase inhibitory activity, and a surfactant.
  • the lysis buffers provided herein can include a buffer, such as Tris or Tris base, HEPES, CHAPS, or the like, at pH 6.0 to 9.0 for a range of temperatures such as 5 °C to 40 °C.
  • the lysis buffers can include a chelator (e.g., EDTA, EGTA or the like), or can be substantially free of a chelator.
  • Anionic oligomers having RNase inhibitory activity Several anionic oligomers having RNase inhibitory activity are known in the art, and are useful in the embodiments provided herein. Non-limiting examples of anionic oligomers having RNase inhibitory activity useful in the embodiments described herein include Poly(vinylphosphonic acid), Heparin, Sulfated cellulose, Sulfated nitro-carboxymethyl cellulose, Sulfated amylose, Sulfated amylopectin, Sulfated pectic acid, Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Fucoidan, Poly(2-acrylamindo-2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly-p,p-dioxy-dibenzyl phosphate, Poly-p,p- dioxydiphenyldimethyl metaphosphate, Polyaspartic acid, Polyglutamic acid
  • Anionic oligomers can be present in the lysis mixture in amounts ranging from about 0 ug/mL to about 300 ug/mL. In some embodiments, the anionic oligomer is present in the lysis mixture in amounts ranging from about 20 ug/mL to about 280 ug/mL. In some embodiments, the anionic oligomer is present in the lysis mixture in amounts ranging from about 40 ug/mL to about 250 ug/mL. In some embodiments, the anionic oligomer is present in the lysis mixture in amounts ranging from about 50 ug/mL to about 200 ug/mL.
  • the anionic oligomer is present in the lysis mixture in amounts ranging from about 50 ug/mL to about 150 ug/mL. In some embodiments, the anionic oligomer is present in the lysis mixture in amounts ranging from about 50 ug/mL to about 125 ug/mL. In some embodiments, the anionic oligomer is present in the lysis mixture in amounts ranging from about 60 ug/mL to about 100 ug/mL. In some embodiments, the anionic oligomer is present in the lysis mixture in amounts ranging from about 70 ug/mL to about 90 ug/mL.
  • the anionic oligomer having RNase inhibitory activity is selected from the group consisting of: Poly(vinyl sulfonic acid), Poly(4-styrenesulfonic acid), and Dextran sulfate, for example.
  • the anionic oligomer is present at about 20 ug/mL, 25 ug/mL, 30 ug/mL 35 ug/mL 40 ug/mL, 45 ug/mL 50 ug/mL, 55 ug/mL, 60 ug/mL, 65 ug/mL, 70 ug/mL, 75 ug/mL, 80 ug/mL, 85 ug/mL, 87.5 ug/mL, 90 ug/mL, 95 ug/mL, 100 ug/mL, 105 ug/mL, 110 ug/mL, 115 ug/mL, 120 ug/mL, 125 ug/mL, 130 ug/mL, 135 ug/mL, 140 ug/mL, 145 ug/mL, 150 ug/mL, 155 ug/mL, 160 u
  • the lysis buffer comprises a surfactant.
  • the surfactant is provided at a concentration that has low or no emission at the emission wavelengths of commonly used RNA-or DNA-detectable labels (e.g., between about 300 nm and 750 nm), and wherein the concentration is lysis-effective.
  • the surfactant is REACH compliant.
  • Various surfactants such as cationic surfactants, anionic surfactants, non-ionic surfactants, zwitterionic surfactant, or any combination thereof are known in the art and can be used in the lysis buffers provided herein.
  • the lysis buffer can include a cationic surfactant such as cetyl trimethylammonium bromide (CTAB), cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2- hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxy ethylcellulose ethoxylate, polyquaternium-10, and hexadecyl-trimethylammoniumchloride (HTAC), or any combination thereof.
  • CTAB cetyl trimethylammonium bromide
  • CTAC cetyl trimethylammonium chloride
  • CPC cetylpyridinium chloride
  • HTAC hexadecyl-trimethylammoniumchloride
  • the lysis buffer can include an anionic surfactant.
  • Anionic surfactants useful in the lysis buffers provided herein include, but are not limited to sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxycholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, ammonium laurcth sulfate, or any combination thereof.
  • the lysis buffer can include a non-ionic surfactant, such as for example, one or more of Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15- S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Tergitol NP-30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S- 20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, Pluronic F-127, Synperonic® F 108, Synperonic® PE P105, ECOSURFTM EH-9, EcosurfTM SA-4, EcosurfTM SA-9, EcosurfTM EH-6, EcosurfTM EH
  • the lysis buffer can include one or more zwitterionic surfactants, such as cocamidopropyl betaine (CAPB), CHAPS (3-((3-choIamidopropyI) dimethylammonio)-l -propanesulfonate, CHAPSO (3--([3-
  • a lysis buffer provided herein includes a non-ionic surfactant is selected from the group consisting of Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-1 1 , Tergitol NP-13, Tergitol NP-50, Tergitol NP-30, Tergitol NP-15, Tergitol NP-40, EcosurfTM SA-4, EcosurfTM SA-9, EcosurfTM EH-6, EcosurfTM EH-3, Saponin, EcosurfTM SA-7, Poloxamer 188, Tween® 20, Tween® 60, Tween® 65, Triton X-45TM, Triton X-114TM, Triton X-102TM, Brij® 35, Brij® 58, Brij® L23, Brij® S10, Tergitol NP-8, Tergitol NP-8, Tergit
  • the concentration of surfactant present in the lysis buffer can be sufficient to lyse the majority of the cells, virus, fungi, or the like in the sample.
  • the concentration of surfactant can be such that greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99% of the cells, virus, fungi or the like are lysed in the sample.
  • various methods can be used to determine the percentage of cells, viruses, fungi or the like lysed.
  • propidium iodide as described in the following: www.bmglabtech.com/en/application-notes/high-throughput-method-for- dynamic-measurements-of-cellular-viability-using-a-bmg-labtech-microplate-reader.
  • Lysis-effective concentrations of Tergitol surfactants range from 0.001% to 10% (v/v) or more. Accordingly, a lysis effective concentration of Tergitol can be from 0.05% to 5%, e.g., range from 0.05% to 3%, from 0.05% to 1%, from 0.05% to 0.5%, from 0.05% to 0.3% or more.
  • a DNase' can optionally include preparing RNA from samples.
  • a DNase is optionally present in the lysis buffer, or added to either the sample or the lysis mixture.
  • the DNase is a heat-labile double-strand specific DNase (HL-dsDNase).
  • HL-dsDNase heat-labile double-strand specific DNase
  • the HL-dsDNase is heat inactivated at 55 °C. Accordingly, in RT-qPCR reactions, which generally include inactivation of reverse transcriptase, the HL-dsDNase would be simultaneously inactivated.
  • lysis mixtures described herein are substantially free of a chelator.
  • Common chelators, such as EDTA has been found herein to interfere with deoxyribonuclease activity at 1 mM. Therefore, lysis mixtures provided herein are substantially free of a chelator, have less than about 0.1 mM chelator, have less than about 0.2 mM chelator, have less than about 0.5 mM or have less than 1 mM chelator.
  • lysis mixtures include a chelator.
  • the lysis buffer includes one or more salts, such as alkaline metal salts (e.g, calcium and/or magnesium salts).
  • lysis buffers provided herein can include a calcium salt in concentrations ranging from 0 mM to 2.5 mM.
  • a calcium salt is present in the lysis buffer in concentrations ranging from about 0.25 mM to about 2.5 mM.
  • a calcium salt is present in the lysis buffer in concentrations ranging from about 0.25 mM to about 2.0 mM.
  • a calcium salt is present in the lysis buffer in concentrations ranging from about 0.25 mM to about 1.5 mM.
  • a calcium salt is present in the lysis buffer in concentrations ranging from about 0.25 mM to about 1.0 mM.
  • the calcium salt can be any calcium salt, including but not limited to calcium chloride, calcium bromide, calcium acetate, calcium formate, calcium sulfate, or calcium phosphate, for example.
  • lysis buffers provided herein can include CaCh is present at about 0 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, or 2.5 mM or any range of concentrations therebetween.
  • MgCh is present in the lysis buffer in concentrations ranging from 0 mM to 15.0 mM.
  • MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 15.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 12.5 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 10.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 7.5 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 5.0 mM.
  • MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 4.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 3.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 2.0 mM. In certain embodiments, the MgCh is present at about 0.1 mM, 0.2 mM, 0.5 mM, 1 .0 mM,
  • Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 10 -100 ug/mL PVSA, 10.0-50.0 mM Tris pH 7.5, 1.0-10.0 mM; MgCh, 0.25-2.0 mM; CaCh; and from 0.05% to 4.0% Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, ECOSURFTM EH-9, EcosurfTM SA-4, EcosurfTM SA-9, EcosurfTM EH-6, EcosurfTM EH-3, Brij® 35, Brij® 58, Brij® L23, Brij® S10, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Gly
  • Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 10 -100 ug/mL PVSA, 10.0-50.0 mM Tris pH 7.5, 1.0-10.0 mM; MgCh, 0.25-2.0 mM; CaCh; and from 0.05% to 4.0% Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, ECOSURFTM EH-9, EcosurfTM SA-4, EcosurfTM SA-9, EcosurfTM EH-6, EcosurfTM, or any combination thereof in nuclease free water.
  • the lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.
  • Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 10 -100 ug/mL PVSA, 10.0-50.0 mM Tris pH 7.5, 1.0-10.0 mM; MgCh, 0.25-2.0 mM; CaCh; and from 0.05% to 4.0% sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, ammonium laureth sulfate, or any combination thereof in nuclease free water.
  • the lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.
  • Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 10-100 ug/mL PVSA, 10.0-50.0 mM Tris pH 7.5, 1.0-10.0 mM; MgCh, 0.25-2.0 mM; CaCh; and from 0.05% to 2.0% Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, ammonium laureth sulfate, or any combination thereof in nuclease free water.
  • the lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.
  • Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 50-100 ug/mL PVSA, 10.0-35.0 mM Tris pH 7.5, 1.0-7.5 mM; MgCh, 0.25-1.5 mM; CaCh; and from 0.05% to 1.5% Ursodeoxycholic acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxy cholic acid, sodium stearate, or any combination thereof in nuclease free water.
  • the lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.
  • Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 50-80 ug/mL PVSA, 15.0-25.0 mM Tris pH 7.5, 1.0-4.5 mM; MgCh, 0.25-1.0 mM; CaCh; and from 0.05% to 1.0% Chenodeoxycholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, or any combination thereof in nuclease free water.
  • the lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty -four months.
  • Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 50-80 ug/mL PVSA, 15.0-25.0 mM Tris pH 7.5, 1.0-3.0 mM; MgCh, 0.25-1.0 mM; CaCh; and from 0.05% to 0.75% Taurocholic Acid, Taurodeoxycholic acid, Deoxycholic acid, or any combination thereof in nuclease free water.
  • the lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.
  • Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 50-80 ug/mL PVSA, 15.0-25.0 mM Tris pH 7.5, 1.0-3.0 mM; MgCh, 0.25-0.75 mM; CaCh; and from 0.05% to 0.5% Taurocholic Acid, Taurodeoxycholic acid, Deoxycholic acid, or any combination thereof in nuclease free water.
  • the lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.
  • the lysis mixtures and cell lysates produced therefrom provided herein can be used in any number of downstream reactions and processes.
  • the cell lysates can be used in RT-qPCR reactions, single-cell analysis reactions (e.g., RNA-seq and the like), next-generation sequencing (NGS) reactions, multiplex amplification reactions (e.g., AMPLISEQ®), Northern Blotting, in vitro transcription, or the like.
  • NGS next-generation sequencing
  • AMPLISEQ® multiplex amplification reactions
  • Northern Blotting in vitro transcription, or the like.
  • Detection of RNA or DNA or a surrogate thereof includes detection means using emission by an emitter that is representative of the RNA or DNA.
  • RNA present in a cell lysate produced by the methods described herein is detected in situ by adding or mixing at least a portion of the lysis mixture with a composition comprising reverse transcriptase to produce an RT product, e.g., that comprises cDNA.
  • the RT product provides a surrogate of the RNA that can be detectable.
  • any reverse transcriptase known to those of ordinary skill in the art can be used such as, for example, MMLV-RT (murine maloney leukemia virus-reverse transcriptase), avian myelogenous virus reverse transcriptase (AMV-RT), human immunodeficiency virus (HIV)-RT and the Tth DNA polymerase which has reverse transcriptase activity if Mn ++ is provided.
  • MMLV-RT murine maloney leukemia virus-reverse transcriptase
  • AMV-RT avian myelogenous virus reverse transcriptase
  • HV human immunodeficiency virus
  • Tth DNA polymerase which has reverse transcriptase activity if Mn ++ is provided.
  • the HL-dsDNase is heat inactivated during the downstream processes of the RT protocol.
  • a positive control RNA can be added to the lysis buffer or the cell lysate.
  • Amplification refers to a process that results in an increase in the copy number of a molecule or set of related molecules.
  • amplification means the production of multiple copies of the target nucleic acid, a surrogate of a target nucleic acid, or a portion thereof.
  • Amplification can encompass a variety of chemical and enzymatic processes such as a polymerase chain reaction (PCR), a strand displacement amplification reaction, a transcription mediated amplification reaction, an isothermal amplification reaction, or a nucleic acid sequence-based amplification reaction, for example.
  • PCR polymerase chain reaction
  • the amplification products can be detected or can be separated from at least one other component of the amplification mixture based on their molecular weight or length or mobility prior to detection.
  • PCR includes introducing a molar excess of two or more extendable oligonucleotide primers to a reaction mixture comprising the lysis mixture where the primers hybridize to opposite strands of a DNA, RNA, or RNA surrogate.
  • the reaction mixture is subjected to a program of thermal cycling in the presence of a DNA polymerase, resulting in the amplification of the DNA or RNA surrogate sequence flanked by the primers.
  • Reverse transcriptase PCR is a PCR reaction that uses an RNA template and a reverse transcriptase, or a polypeptide having reverse transcriptase activity, to first generate a single stranded DNA molecule prior to the multiple cycles of DNA-dependent DNA polymerase primer elongation as cited above.
  • Methods for a wide variety of PCR applications arc widely known in the art, and described in many sources, for example, Ausubel et al. (eds.), Current Protocols in Molecular Biology, Section 15, John Wiley & Sons, Inc., New York (1994).
  • sequence-specific primers are well known to persons of ordinary skill in the art. Detailed descriptions of primer design that provide for sequence- specific annealing can be found, among other places, in Diffenbach and Dveksler, PCR Primer, A Laboratory Manual, Cold Spring Harbor Press, 1995, and Kwok etal. (Nucl. Acid Res. 18:999- 1005, 1990).
  • the sequence- specific portions of the primers are of sufficient length to permit specific annealing to complementary sequences, as appropriate.
  • a primer does not need to have 100% complementarity with a primer- specific portion for primer extension to occur.
  • a primer can be detectably labeled such that the label is detected by spectroscopy.
  • a primer pair is sometimes said to consist of a "forward primer” and a "reverse primer,” indicating that they are initiating nucleic acid polymerization in opposing directions from different strands of a duplex template.
  • a primer as set forth herein can comprise a universal priming sequence.
  • the term “universal primer” refers to a primer comprising a universal sequence that is able to hybridize to all, or essentially all, potential target sequences in a multiplexed reaction.
  • the term “semi-universal primer” refers to a primer that is capable of hybridizing with more than one (e.g., a subset), but not all, of the potential target sequences in a multiplexed reaction.
  • the terms “universal sequence,” “universal priming sequence” or “universal primer sequence” or the like refer to a sequence contained in a plurality of primers, where the universal priming sequence that is found in a target is complementary to a universal primer.
  • a passive reference dye ROXTM can be included in PCR reactions to provide an internal reference to which the reporter-dye signal can be normalized during data analysis. Normalization can be accomplished using Applied Biosystems’ Design and Analysis software.
  • single- stranded amplification products can be generated by methods including, without limitation, asymmetric PCR, asymmetric reamplification, nuclease digestion, and chemical denaturation.
  • single- stranded sequences can be generated by combining at least one first primer or at least one second primer from a primer set, but not both, in an amplification reaction mixture, or by transcription, for example, when a promoter-primer is used in a first amplification mixture, a second amplification mixture, or both.
  • Polymerase refers to a polypeptide that is able to catalyze the addition of nucleotides or analogs thereof to a nucleic acid in a template dependent manner, for example, the addition of deoxyribonucleotides to the 3 ’ -end of a primer that is annealed to a nucleic acid template during a primer extension reaction.
  • Nucleic acid polymerases can be thermostable or thermally degradable.
  • thermostable polymerases include, but are not limited to, polymerases isolated from Thermus aquaticus, Thermus thermophilus, Pyrococcus woesei, Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritima.
  • Suitable thermodegradable polymersases include, but are not limited to, E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, and others.
  • examples of other polymerizing enzymes that can be used in the methods described herein include but are not limited to T7, T3, SP6 RNA polymerases; and AMV, M-MLV and HIV reverse transcriptases.
  • polymerases include, but are not limited to AMBION’S SUPERTAQ®, TAQFS®, AMPLITAQ® CS (Applied Biosystems), AMPLITAQ® FS (Applied Biosystems), KENTAQ1® (AB Peptide, St. Louis, Mo.), TAQUENASE® (Scien Tech Corp., St.
  • reverse transcription and/or amplification is optionally followed by additional steps, for example, but not limited to, labeling, sequencing, purification, isolation, hybridization, size resolution, expression, detecting and/or cloning.
  • one or both reverse transcription and/or PCR primers can comprise a label, such as, for example, a fluorophore.
  • a label can facilitate detection of an amplification product comprising a labeled PCR primer.
  • biotinylated strands can be captured, separated, and detected.
  • Multiplex Assays refers to reverse transcription and/or PCR reactions that use more than two primers in a single reaction and at the same time so that more than one different amplified product is produced and detected. For example, more than two pair of amplification primers are contacted at the same time and/or in the same solution. Several target RNAs or DNAs can be detected simultaneously using multiplex assays.
  • Real-time PCR refers to the detection and quantitation of an RNA, a DNA or a surrogate thereof in a sample.
  • the amplified segment or “amplicon” can be detected using a 5'-nuclease assay, particularly the TAQMAN® assay as described by e.g., Holland et al. Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991); and Heid etal. ⁇ Genome Research 6:986-994, 1996).
  • a TAQMAN® nucleotide sequence to which a TAQMAN® probe binds can be designed into the primer portion, or known to be present in an RNA or a DNA of a sample.
  • T m refers to the melting temperature (temperature at which 50% of the oligonucleotide is a duplex) of an oligonucleotide determined experimentally or calculated using the nearest-neighbor thermodynamic values of Breslauer et al. (Proc. Natl. Acad. Sci. USA 83:3746 3750, 1986) for DNA or Freier etal. (Proc. Natl. Acad. Sci. USA 83:9373-9377, 1986) for RNA.
  • the T m of the TAQMAN® probe is about 10 degrees above the T m of amplification primer pairs.
  • Amplification primer sequences and double dye-labeled TAQMAN® probe sequences can be designed using PRIMER EXPRESSTM (Version 1.0, Applied Biosystems, Foster City, CA) or mFOLDTM software (now UNIFoldTM) (IDT, San Jose, CA).
  • a TAQMAN® probe When a TAQMAN® probe is hybridized to RNA, DNA, or a surrogate thereof, the 5'- exonuclease activity of a thermostable DNA-dependent DNA polymerase such as SUPERTAQ® (a Taq polymerase from Thermus aquaticus, Ambion, Austin, TX) digests the hybridized TAQMAN® probe during the elongation cycle, separating the fluor from the quencher. The reporter fluor dye is then free from the quenching effect of the quencher moiety resulting in a decrease in FRET and an increase in emission of fluorescence from the fluorescent reporter dye. One molecule of reporter dye is generated for each new molecule synthesized, and detection of the free reporter dye provides the basis for quantitative interpretation of the data.
  • a thermostable DNA-dependent DNA polymerase such as SUPERTAQ® (a Taq polymerase from Thermus aquaticus, Ambion, Austin, TX) digests the hybridized TAQMAN® probe during the elongation cycle,
  • the amount of fluorescent signal is monitored with each cycle of PCR. Once the signal reaches a detectable level, it has reached the "threshold or cycle threshold (Ct).”
  • Ct threshold or cycle threshold
  • a Anorogenic PCR signal of a sample can be considered to be above background if its Ct value is at least 1 cycle less than that of a no-template control sample.
  • the term “Ct” represents the PCR cycle number when the signal is first recorded as statistically significant. Thus, the lower the Ct value, the greater the concentration of nucleic acid target.
  • the Auorescent signal should double if there is no inhibition of the reaction and the reaction was nearly 100% efficient with purified nucleic acid.
  • Detection method embodiments using a TAQMAN® probe sequence comprise combining the lysate mixture or the reverse transcribed mixture with PCR reagents, including a primer set having a forward primer and a reverse primer, a DNA polymerase, and a Auorescent detector oligonucleotide TAQMAN® probe, to form an amplification reaction mixture; subjecting the amplification reaction mixture to successive cycles of amplification to generate a fluorescent signal from the detector probe; and quantitating the nucleic acid presence based on the fluorescent signal cycle threshold of the amplification reaction.
  • Protocols and reagents for means of carrying out further 5'-nuclease assays are well known to one of skill in the art, and are described in various sources.
  • 5'-nuclease reactions and probes are described in U.S. Patent No’s. 6,214,979 issued Apr. 10, 2001; 5,804,375 issued Sep. 8, 1998; 5,487,972 issued Jan. 30, 1996; and 5,210,015 issued May 11, 1993, all to Gelfand et al.
  • a detection method can utilize any probe that can detect a nucleic acid sequence.
  • a detection probe can be, for example, a TAQMAN® probe described supra, a stem-loop molecular beacon, a stemless or linear beacon, a PNA MOLECULAR BEACONTM, a linear PNA beacon, non-FRET probes, SUNRISE® /AMPLIFLUOR® probes, stem-loop and duplex SCORPIONTM probes, bulge loop probes, pseudo knot probes, cyclicons, MGB ECLIPSETM probe, a probe complementary to a ZIPCODETM sequence, hairpin probes, peptide nucleic acid (PNA) light-up probes, selfassembled nanoparticle probes, and ferrocene-modified probes as known by one of ordinary skill in the art.
  • PNA peptide nucleic acid
  • a detection probe having a sequence complementary to a detection probe hybridization sequence such as a ZIPCODETM sequence, a Iluorphore and a mobility modifier can be, for example, a ZIPCHUTETM probe supplied commercially by Applied Biosystems (Foster City, CA).
  • label or Reporter refers to a moiety or property that allows the detection of that with which it is associated and, for use herein, has emission spectra at between and including 300 nm to 750 nm.
  • the emission spectra is at less than about 499 nm such as for blue emitters such as certain Alexa Fluor emitters, Cascade Blue, Pacific Blue, Biosearch BlueTM, ATTOTM 390, ATTOTM 425, and Cyan 500; at 500 nm to 549 nm emitters such as for green emitters such as certain Alexa Fluor emitters, BODIPY FL, fluorescein (FITC), cyanine 2, Catskill Green, 5-FAM, 6-FAM, succinimidyl ester, JOE, MFP488, the Oregon Green emitters, TETTM, ATTOTM 488, Rhodamine GreenTM-X, LC® CYAN 500, LC® Fluo, ATTOTM 465, and ATTOTM' 495; at 550 nm to 584 nm emitters such as yellow emitters such as certain Alexa Fluor emitters, Cyanine 3, HEXTM, NED, R-Phycoerythrin (R-PE), 5-TAMRA, TRI
  • the label can be attached covalently or non-covalently to an RNA product, to a DNA product, or to a surrogate thereof such as an amplicon thereof.
  • Commonly used labels include dyes that are negatively charged, such as dyes of the fluorescein family including, e.g. EAM, HEX, TET, JOE, NAN and ZOE; or dyes that are neutral in charge, such as dyes of the rhodamine family including, e.g., Texas Red, ROXTM, R110, R6G, and TAMRA; or dyes that are positively charged, such as dyes of the cyanine family including e.g., Cyl, Cy3, Cy5, Cy5.5 and Cy7.
  • FAM, HEX, TET, JOE, NAN, ZOE, ROXTM, R110, R6G, and TAMRA are available from, e.g., Perkin-Elmer, Inc. (Wellesley, MA); Texas Red is available from, e.g., Molecular Probes, Inc. (Eugene, OR); and Cy2, Cy3, Cy5, Cy5.5 and Cy7, and are available from, e.g., Amersham Biosciences Corp. (Piscataway, NJ).
  • the fluorescer molecule is a fluorescein dye and the quencher molecule is a rhodamine dye.
  • a label or reporter can comprise both a fluorophore and a fluorescence quencher.
  • the fluorescence quencher can be a fluorescent fluorescence quencher, such as the fluorophore TAMRA, or a non-fluorescent fluorescence quencher (NFQ), for example, a combined NFQ- minor groove binder (MGB) such as an MGB ECLIPSETM minor groove binder supplied by Epoch Biosciences (Bothell, WA) and used with TAQMANTM probes (Applied Biosystems).
  • MGB combined NFQ- minor groove binder
  • MGB NFQ- minor groove binder supplied by Epoch Biosciences (Bothell, WA) and used with TAQMANTM probes (Applied Biosystems).
  • the fluorophore can be any fluorophore that can be attached to a nucleic acid, such as, for example, FAMTM, HEXTM, TETTM, JOETM, NAN, ZOE, Texas Red, ROXTM, R110, R6G, TAMRATM, Cy2, Cy3, Cy5, Cy5.5 and Cy7 as cited above as well as VIC, NED, LIZ, ALEXA, Cy9, and dR6G.
  • Labels include black hole quenchers (BHQ) (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch).
  • Labels can also comprise sulfonate derivatives of fluorescein dyes, phosphoramiditc forms of fluorescein, phosphoramiditc forms of CY5 (available for example from Amersham), intercalating labels such as ethidium bromide, and SYBRTM Green I and PICOGREENTM (Molecular Probes).
  • RNA, DNA, or a surrogate thereof comprise use of a promoter sequence or a complement thereof and the method includes combining the RNA, DNA, or a surrogate thereof with PCR reagents, including at least one primer set and a DNA polymerase, to form a first amplification reaction mixture subjecting the first amplification reaction mixture to at least one cycle of amplification to generate a first amplification product comprising the promoter sequence; combining the first amplification product with an RNA polymerase and a ribonucleoside triphosphate solution comprising at least one of rATP, rCTP, rGTP, rUTP, or aminoallyl-rUTP to form a transcription reaction mixture; incubating the transcription reaction mixture under appropriate conditions to generate an RNA transcription product; and detecting presence of the target nucleic acid by detection of the RNA transcription product or a portion thereof.
  • the polymerase is reverse transcriptase.
  • RNA polymerases include T7, T3, or SP6 RNA polymerase and exemplary promoters include the T7, T3, or SP6 promoters.
  • the RNA transcription product or a portion thereof can be detected using, for example, the aminoallyl-rUTP which is available for coupling to a succinimide ester label for detection.
  • Enzymatically Active Mutants or Variants Thereof refers to a polypeptide derived from the corresponding enzyme that retains at least some of the desired enzymatic activity.
  • Enzymatically active mutants or variants include, for example, fragments, recombinantly expressed fragments, naturally-occurring mutants, mutants generated using mutagens, genetically engineered mutants, mutants due to amino acid insertions or deletions or due to nucleic acid nonsense, missense, or frameshift mutations, reversibly modified enzymes, splice variants, polypeptides having modifications such as altered glycosylation, disulfide bonds, hydroxyl side chains, and phosphate side chains, or crosslinking, and the like. Protocols for measuring enzymatic activity using an appropriate assay are known to one of ordinary skill in the art.
  • Cell lysates provided herein are useful for any method of detection of nucleic acid that uses a dye that has a detectable emission.
  • a dye or label that fluoresces in the 500nm to 615 nm range such as used in PCR, RT-PCR, qRT-PCR, siRNA-mediated gene knockdown, high-throughput assessment of any kind particularly in 96-well or 384- well plates is envisioned for use herein.
  • Samples can be processed directly in culture plates, minimizing sample handling and the potential for sample loss or transfer error.
  • the cell lysis protocol in 384-well plates is readily automated on robotic platforms.
  • cDNA can then be synthesized directly from the lysate using the Superscript IV VILO RNase and Applied Biosystems QuantStudioTM 5 Real Time PCR instrument.
  • Custom libraries of Silencer® Pre-designed siRNAs and TAQMAN® Gene Expression Assays plated to specification in 384-well plates can be obtained directly from the manufacturer (Applied Biosystems). Processes provided by the teachings herein ensure high-throughput processing, efficient use of reagents and instruments, a minimal amount of hands-on time, and accurate and reliable results.
  • Kits A "kit,” as used herein, refers to a combination of items for performing a sample preparation method as set forth herein.
  • Kits provided herein can include a lysis buffer, as described herein above.
  • kits provided herein can include a lysis buffer that includes a surfactant (e.g., Tergitol 15-S-9, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, ammonium laureth sulfate, EcosurfTM SA-4, EcosurfTM SA-9, Ecosurf 1M EH-6, EcosurfTM EH-3, Saponin, Ecosurf TM SA-7, Polox
  • the lysis buffer includes a DNase, such as a HL-dsDNase.
  • the DNase can be present in the buffer, or alternatively provided in a separate container from the lysis buffer.
  • the lysis buffer includes a RNase inhibitor protein.
  • the RNase inhibitor protein can be present in the buffer, or alternatively provided in a separate container from the lysis buffer.
  • the lysis buffer is substantially free of a chelator. Components of kits may be packaged together or separately as desired for the processes described herein.
  • Kits can further include reagents for reverse transcription, such as reverse transcriptase, a reverse primer, dNTPs or a reverse transcriptase buffer, or can further comprise reagents for PCR, such as a DNA polymerase, or dNTPs, for example.
  • reagents for reverse transcription such as reverse transcriptase, a reverse primer, dNTPs or a reverse transcriptase buffer
  • reagents for PCR such as a DNA polymerase, or dNTPs, for example.
  • kits can include probes, e.g., for the detection of target nucleic acids.
  • a detector probe such as a 5 ’-nuclease probe such as a TAQMAN® probe, an RNA or a DNA control nucleic acid, reagents for sample collection, an RNA polymerase or an enzymatically active mutant or variant thereof, or ribonucleotides rATP, rCTP, rGTP, rUTP, or aminoallyl-rUTP.
  • Kits can also include enzymes such as Thermus sp. ZO5 polymerase or Thermits thermophilus polymerase.
  • the liquid solution comprises an aqueous solution that can be a sterile aqueous solution.
  • the components of the kit can be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent.
  • the solvent can also be provided in another container means.
  • the container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the solutions are placed, and in some embodiments, suitably aliquoted.
  • the kits can also comprise a further container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
  • kits can also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions can include variations that can be implemented.
  • Methods of Enhancing DNase Activity Certain methods provided herein are based, in part, on the surprising discovery that the inclusion of an anionic oligomer with RNase inhibiting properties in the presence of a DNase digestion reaction enhances the efficiency of DNA digestion by the DNase. Accordingly, provided herein are methods and compositions to enhance the efficiency of DNA digestion by DNase. [0118] In one aspect, these methods can include the steps of providing a sample containing double-stranded DNA. The skilled artisan will appreciate that the methods arc useful for digestion of double-stranded DNA from any source, including genomic DNA (gDNA), plasmid DNA, products of amplification reactions, and the like.
  • gDNA genomic DNA
  • plasmid DNA products of amplification reactions, and the like.
  • the sample is contacted with a double-strand specific DNase (e.g., a HL-dsDNase) and an anionic oligomer having RNase inhibiting properties to produce a digestion reaction, and the digestion reaction can be incubated at a temperature for a period of time.
  • a double-strand specific DNase e.g., a HL-dsDNase
  • an anionic oligomer having RNase inhibiting properties to produce a digestion reaction
  • the digestion reaction can be incubated at a temperature for a period of time.
  • the anionic oligomer having RNase inhibitor properties can be Poly(vinylphosphonic acid), Poly(vinyl sulfonic acid), Fucoidan, Poly(2-acrylamindo-2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly(4-styrenesulfonic acid), Polyaspartic acid, Polyglutamic acid, Polyacrylic acid, Poly(methacrylic acid), Poly(maleic acid), Dextran sulfate, or combinations thereof.
  • the anionic oligomer having RNase inhibiting properties is Poly(vinyl sulfonic acid) (PVSA).
  • the anionic oligomer can be present in an amount between about 5 ug/ml to about 300 ug/ml in the digestion reaction. For example, about 20 ug/mL, 25 ug/mL, 30 ug/mL 35 ug/mL 40 ug/mL, 45 ug/mL 50 ug/mL, 55 ug/mL, 60 ug/mL, 65 ug/mL, 70 ug/mL, 75 ug/mL, 80 ug/mL, 85 ug/mL, 87.5 ug/mL, 90 ug/mL, 95 ug/mL, 100 ug/mL, 105 ug/mL, 110 ug/mL, 115 ug/mL, 120 ug/mL, 125 ug/mL, 130 ug/mL, 135 ug/mL, 140 ug/mL,
  • the DNase e.g., HL-dsDNase can be present in the digestion reaction between about lU/mL to about 500U/mL.
  • the methods can include contacting the sample with 10 U/mL, 15 U/mL, 20 U/mL, 25 U/mL, 30 U/mL, 35 U/mL, 40 U/mL, 45 U/mL, 50 U/mL, 55 U/mL, 60 U/mL, 65 U/mL, 70 U/mL, 75 U/mL, 80 U/mL, 85 U/mL, 90 U/mL, 95 U/mL, 100 U/mL, 125 U/mL, 150 U/mL, 175 U/mL, 200 U/mL, or greater, or any amount in between.
  • the anionic oligomer and the DNase can be provided in a digestion reaction buffer, or added to the digestion reaction directly. Accordingly, some embodiments provide a method wherein the sample containing double- stranded DNA is contacted with a reaction buffer that includes both an anionic oligomer that has RNase inhibiting properties (e.g., PVSA) and a DNase (e.g., HL-dsDNase).
  • a reaction buffer that includes both an anionic oligomer that has RNase inhibiting properties (e.g., PVSA) and a DNase (e.g., HL-dsDNase).
  • the sample containing double- stranded DNA can be contacted with a reaction buffer that includes an anionic oligomer that has RNase inhibiting properties (e.g., PVSA) to produce a first mixture, and the DNase (e.g., HL-dsDNase) is added directly to the first mixture, to produce a reaction mixture.
  • the sample containing double-stranded DNA is contacted with a reaction buffer that includes a DNase (e.g., a HL-dsDNase) to produce a first mixture, and the anionic oligomer that has RNase inhibiting properties is added directly to the first mixture to produce a reaction mixture.
  • the digestion reaction can proceed in a digestion reaction buffer that contains, inter alia, salts, buffering agents, and other components that are typically present in a DNase digestion reaction.
  • the digestion reaction can be incubated between about 15 °C to 40 °C, or about 16 °C to 28 °C or about 19 °C to 26 °C, or about 19 °C to 25 °C, or about 22 °C to 25 °C, or at ambient temperature, or about 15 °C, 16 °C, 17 °C, 18 °C, 19 °C,
  • the reaction mixture remains at substantially the same temperature during the incubation time.
  • “Substantially the same temperature” generally refers to an isothermal process of holding the temperature relatively constant during the incubation time, for certain embodiments described herein, means ambient temperature which temperature may change during the day or from lab to lab. An isothermal process is particularly amenable for high throughput analyses.
  • the incubation temperature is such that the DNase, e.g., HL-dsDNase, is not inactivated (e.g., below 50°C).
  • the reaction mixture is incubated for a period of time.
  • digestion reaction mixtures can be incubated at 16 °C to 28 °C for 2 minutes to about 60 minutes, about 2 minutes to about 20 minutes, about 3 minutes to about 15 minutes, about 4 minutes to about 10 minutes or about 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, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes 34 minutes 35 minutes 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes 42 minutes 43 minutes, 44 minutes 45 minutes, 46 minutes 47 minutes, 48 minutes 49 minutes, 50 minutes, 51 minutes, 52 minutes 53 minutes 54 minutes 55 minutes 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, or longer, or any time in between.
  • lysis mixtures can be held on ice, or incubated at 4 °C for 15 minutes to 12 hours or longer, e.g., 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer, or any time in between.
  • the presence of the anionic oligomer having RNase inhibiting properties to the digestion reaction can increase the digestion of the double- stranded DNA in a sample by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 405, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, or more, compared to similar samples processed under the same conditions, but in the absence of the anionic oligomer.
  • Example 3 below provides a non-limiting, exemplary method of determining the amount of digestion of double-stranded DNA.
  • kits for the digestion of double-stranded DNA, and kits containing the same.
  • the kits contain a digestion reaction buffer that includes both an anionic oligomer having RNase inhibiting properties and a DNase, e.g., a HL-dsDNase, an optionally salts, buffers, and the like.
  • the kits contain a digestion reaction buffer that includes an anionic oligomer having RNase inhibiting properties, and optionally salts, buffers, and the like.
  • Such kits can contain a DNase in a separate container.
  • the kits contain a digestion reaction buffer that includes the DNase, and optionally salts, buffers, and the like.
  • Such kits can contain an anionic oligomer having RNase inhibiting properties in a separate container.
  • Exemplary digestion reaction buffers include, for example, salts (e.g., magnesium and/or calcium salts), buffers (e.g, Tris or Tris base buffers, HEPES, CHAPs, or the like), and an anionic oligomer having RNase inhibiting properties.
  • CaCh is present at about 0 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, or 2.5 mM or any range of concentrations therebetween.
  • MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 12.5 mM.
  • MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 10.0 mM. In some embodiments, MgCb is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 7.5 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 5.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 4.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 3.0 mM.
  • MgCh is present in the digestion reaction buffer in concentrations ranging from 0 mM to 2.5 mM. In some embodiments, MgCh is present in the digestion reaction buffer in concentrations ranging from about 0.5 mM to about 2.5 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 2.0 mM. In some embodiments, MgCh is present in the digestion reaction buffer in concentrations ranging from about 1.0 mM to about 2.0 mM.
  • the MgCh is present at about 0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 6.5 mM, 7.0 mM, 7.5 mM, 8.0 mM, 8.5 mM, 9.0 mM, 9.5 mM, 10.0 mM, 10.5 mM, l l.O mM, 11.5 mM, 12.0 mM, 12.5 mM, 13.0 mM, 13.5 mM, 14.0 mM, 14.5 mM, 15.0 mM or any range of concentrations there between.
  • the digestion reaction buffers can be substantially free of a chelator.
  • the digestion reaction buffers can contain a chelator.
  • digestion reaction buffers provided herein can be provided in concentrated form, e.g., 2X, 5X, 10X, 20X or the like, and diluted to IX.
  • the digestion reaction buffers provided herein can be provided in lyophilized form, and reconstituted.
  • a lysis solution was prepared that consisted of 10 mM Tris pH 7.5, 5 mM MgCh, 50 ug/mL PVSA, and 0.1% of non-ionic surfactant.
  • the non-ionic surfactants tested included: Tergitol 15-S-9, Tergitol 15- S-12, EcosurfTM EH-9 and Brij 58. 50 pL of each lysis solution or PBS was used to lyse 6,950 HepG2 cells that were suspended in 5 pL of PBS.
  • RNA 1 pL of lysate or purified RNA was used as a template for 1-step RT-qPCR using the Applied BiosystemsTM TaqManTM Fast Virus 1-Step Master Mix and TaqMan Gene Expression Assays targeting the IMPA2 (5’ FAM-labeled TaqMan probe) and ROCK2 (5’ VIC-labeled TaqMan probe) genes.
  • the cell lysate comprised 10% of the total reaction volume. Reactions were run on an Applied Biosystems QuantStudioTM 5 Real-Time PCR Instrument.
  • FIG. 1 Demonstrates that a variety of non-ionic surfactants are effective at lysing human cell cultures.
  • a lysis solution was prepared that consisted of 25 mM Tris pH 7.5, 0.1% Tergitol 15-S-9, 2.5 mM MgCh, 0.5 mM CaCh, and 1U of HL-dsDNase (ArcticZymes). PVSA was supplemented at 75 g/mL or omitted for comparison. 50 pL of each lysis solution was used to lyse 10,000 HeLa cells that were suspended in 5 pL of PBS.
  • the lysate was mixed by pipette 5 times, and then incubated at room temperature over a time course of: 5 minutes, 2 hours, 5 hours, and 20 hours. After each timepoint, cell lysate was added to a SuperScriptTM IV VILO (Invitrogen) reverse transcription reaction. The cell lysate comprised 20% of the total reverse transcription reaction volume. [0135] Reactions were then temperature cycled according to the manufacturer protocol. 2 pL from these reactions were then used as a template for qPCR analysis using the Applied Biosystems TaqMan Fast Advanced Master Mix with a TAQMAN® Gene Expression Assay targeting the PPIA gene (5’ FAM-labeled TaqMan probe).
  • cDNA comprised 20% volume of the qPCR reaction. Reactions were run on an Applied Biosystems QuantStudioTM 5 Real-Time PCR Instrament. Lysis reactions containing PVSA were found to be more stable over time as compared to lysis reactions that did not contain PVSA as indicated by lower Ct values observed for the lysates that contained PVSA at each time point. (FIG. 2).
  • FIG. 2 Provides data demonstrating that the addition of PVSA to the cell lysis buffer helps preserve RNA from being degraded over a 20 hr timecourse.
  • HeLa cells were lysed with lysis buffers containing 75 pg/mL PVSA (white bars) or no PVSA (black bars) and incubated for either 0 hrs, 2 hrs, 5 hrs, or 20 hrs at room temperature.
  • RNA preparations were subjected to gene expression analysis by RT-qPCR using a 5’ FAM-labeled TAQMAN® Gene Expression Assay targeting the PPIA gene. Samples containing PVSA exhibit lower Ct values at each timepoint as compared to samples without PVSA.
  • PVSA HL-dsDNase-mediated digestion of genomic DNA in HeLa cell lysates.
  • a lysis solution was prepared that consisted of 10 mM Tris pH 7.5, 0.1% Tergitol 15-S-9, 5 mM MgCh, 0.5 mM CaCh, and 2U of HL-dsDNase.
  • PVSA was supplemented at concentrations of 0 pg/mL, 50 pg/mL, 75 pg/mL, 87.5 pg/mL, 100 pg/mL and 125 pg/mL.
  • lysis buffers 50 pL of each of these lysis buffers were used to lyse 80,000 HeLa cells suspended in 5 pL of PBS. After applying the lysis buffer to the cells, the lysate was mixed by pipette 5 times, and then incubated at room temperature for 5 minutes. After incubation, the cell lysate was added to a SuperScriptTM IV VILO (Invitrogen) reaction that did not include the reverse transcriptase enzyme. Cell lysate comprised 20% of the total reaction volume.
  • SuperScriptTM IV VILO Invitrogen
  • reactions were then temperature cycled according to the manufacturer protocol. This reaction was then used as a template for qPCR analysis using the Applied Biosystems TAQMAN® Fast Advanced Master Mix with a TAQMAN® Gene Expression Assay targeting the PPTA gene. Reactions were run on an Applied Biosystems QuantStudio 5 Real-Time PCR Instrument. Comparison of the Ct values obtained from the titration of PVSA (FIG.
  • FIG 3 Provides data demonstrating the surprising finding that PVSA enhances the digestion of gDNA by HL-dsDNase in cell lysates. PVSA was supplemented to the cell lysis buffer at the concentrations indicated and gDNA content was analyzed using qPCR with a 5’ FAM-labeled TAQMAN® Gene Expression Assay targeting the PPIA gene.
  • Exemplary non-limiting embodiments of lysis solutions are prepared by obtaining stock solutions of IM Tris-base pH 7.5, IM MgCh, IM CaCh, 20% Tergitol 15-S-9 surfactant, 30% v/v PVSA and nuclease-free water. Stock solutions are diluted to form a lysis solution of Tris pH 7.5, 25 mM; MgCh, 2.5 mM; CaCh, 0.5 mM; Tergitol 15-S-9 surfactant, 0.1%, and 75 pg/mL PVSA in nuclease free water.
  • the pH is adjusted to pH 7.5 +/- 0.1 with HC1 at a temperature of 19 °C-25 °C (a range of pH values is about 7.2 to 8.0.
  • the lysis solution can be stored at room temperature (15 °C to 25 °C), and at 4 °C, and has been found to be stable at 25 °C for at least 20 months.
  • a lysis mixture is prepared by combining the lysis solution with a heat-labile doublestranded deoxyribonuclease (HL-dsDNase) such as a concentration of 20 U/ml (a range of 4 U/ml - 200 U/ml can be used) for those embodiments in which it is desired to remove DNA.
  • HL-dsDNase heat-labile doublestranded deoxyribonuclease
  • the volume of HL-dsDNase added is less than about 1% of the volume of the final lysis reaction. Lysis can be carried out in a 50 pL volume at a pH of 7.5.
  • Certain embodiments of the processes for preparing a sample for nucleic acid analysis are carried out as follows.
  • HL-dsDNase is mixed with lysis solution and the resultant lysis mixture is stored at room temperature.
  • cells are pelleted (-800 x g for 5 min), the media is removed, and the cells are washed with 0.5 mL of 4 °C PBS per 106 cells and re -pelleted. The supernatant is removed, and the cells are resuspended in 4 °C PBS so that 5 pL contains the desired number of cells for one lysis reaction (10— 10 5 cclls/rcaction).
  • Adhered cells in 96- or 384-wcll plates (10 to 100,000 cells) can also be used with this procedure. No centrifugation is required since the cells remain adhered to the plate throughout the washing procedure.
  • Lysis mixture (50 l) is added to the cells and mixed by pipetting or shaking. The lysis reaction is incubated for 5 minutes at room temperature (15 °C -25 °C). After incubation the lysate is ready for downstream nucleic acid analysis, detection and/or amplification and is used within about 60 minutes for high cell inputs (100,000 cells) or 3 hours for lower cell inputs (10,000 - 10 cells). Lysates can also be stored on ice for ⁇ 5 hours or frozen for longterm storage. In some embodiments, the lysates are stable at room temperature for ⁇ 3 hours when the cells are in suspension.
  • a 5-minute lysis time and mixing 5x with a pipette are provided for some embodiments of nucleic acid preparation methods of the present teachings. Temperatures between 15 °C and 25 °C are provided for certain embodiments of isothermal preparation methods. Washing with 0.5 mL of 4 °C PBS per 10 6 cells is acceptable prior to lysis.
  • Nucleic acid analysis, detection and/or amplification can include a reverse transcription step, a real-time PCR reaction, and/or an RNA transcription step comprising use of an RNA polymerase.
  • the sample preparation process provided by teachings herein provides components that minimally interfere with enzymatic activity and detection methods.
  • FIG. 4 Demonstrates the linearity and efficiency of certain sample preparation processes as provided herein using 5’ FAM-labeled TAQMAN® Gene Expression assay (Applied Biosystems) for -actin (ACTB) and CDK4 over 5 logs of cellular input from 10 cells up to 100,000 cells per lysis reaction. The data demonstrate good linearity down to an input of as few as 10 cells.
  • a lysis solution was prepared that consisted of 25 mM Tris pH 7.5, 0.1% Tergitol 15-S-9, 2.5 mM MgCl 2 , 0.5 mM CaCl 2 , 1U of HL-dsDNase (ArcticZymes) and 40 units of RNase Inhibitor Protein (Ambion). Collagenase IV was supplemented at 0.1 units/pL or omitted for comparison.
  • the cell lysate was added to a SuperScriptTM IV VILO (Invitrogen) reaction.
  • Cell lysate comprised 10% of the total reaction volume. Reactions were then temperature cycled according to the manufacturer protocol. This reaction was then used as a template for qPCR analysis using the Applied Biosystems TAQMAN® Fast Advanced Master Mix with a FAM- labeled TAQMAN® Gene Expression Assay targeting the GPI gene and a VIC-labeled TAQMAN® Gene Expression Assay targeting the ACSL3 gene. Reactions were run on an Applied Biosystems QuantStudio 5 Real-Time PCR Instrument. Lysis reactions containing Collagenase IV were found to induce better cell lysis as compared to reactions without Collagenase IV as indicated by lower Ct values observed for the lysates that contained Collagenase IV (FIG. 5).
  • FIG. 5 Provides data demonstrating that the addition of Collagenase IV to the cell lysis buffer helps lyse Primary Hepatocyte cells grown on a Collagen-coated surface and overlayed with Matrigel (Corning) extracellular matrix. Samples containing Collagenase IV in the lysis exhibit lower Ct values as compared to samples without Collagenase IV.
  • a lysis solution was prepared that consisted of 20 mM Tris pH 7.5, 2.5 mM MgCF, 0.5 mM CaC12, 75 ug/mL PVSA, 40U of RNase Inhibitor Protein, 1U of HL-dsDNase and various concentrations of one or more anionic surfactants including Taurodeoxycholic acid and Deoxycholic acid ranging from 0.1% to 0.75%.
  • the cell lysate was added to a SuperScriptTM IV VILO (Invitrogen) reaction.
  • Cell lysate comprised 10% of the total reaction volume. Reactions were then temperature cycled according to the manufacturer protocol. This reaction was then used as a template for qPCR analysis using the Applied Biosystems TAQMAN® Fast Advanced Master Mix with a FAM-labeled TAQMAN® Gene Expression Assay targeting the GPI gene and a VIC-labeled TAQMAN® Gene Expression Assay targeting the ACSL3 gene.
  • cDNA comprised 10% of the reaction volume. Reactions were run on an Applied Biosystems QuantStudio 5 Real-Time PCR Instrument.
  • FIG. 6 Provides data demonstrating that lysis buffers containing anionic surfactants are effective at lysing Human Primary Hepatocyte and the resulting lysates can be added directly to RT-qPCR reactions producing results comparable to purified RNA.
  • Sample preparation processes as provided by teachings herein are compatible with a large number of cell lines.
  • Table 1 provides a listing of cell lines that have been tested.
  • Ct values obtained using lysates as prepared using processes provided herein were found to be essentially equivalent to Ct values obtained with purified RNA.
  • Lysates and purified RNA from 10,000 HeLa cells were prepared in parallel and evaluated with 97 TAQMAN® Gene Expression Assays on an Applied Biosystems QuantStudioTM 12K Flex Real-Time PCR Instrument.
  • the Ct value obtained from the lysates is plotted against the Ct value for the same assay using purified RNA as shown in FIG. 7.
  • FIG. 7 shows that Ct values obtained using lysates as prepared using processes provided herein were found to be essentially equivalent to Ct values obtained with purified RNA.
  • compositions, methods, and kits of the current teachings have been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the current teachings. This includes the generic description of the current teachings with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

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Abstract

L'invention concerne des procédés de préparation d'échantillon pour une analyse in situ d'ARN ou d'ADN, des procédés et des compositions utilisés dans de tels procédés. Les procédés de l'invention permettent de réaliser une préparation d'ADN ou d'ARN et une analyse en aval dans le même tube ou sur une aliquote de l'échantillon préparé sans centrifugation ni purification supplémentaire. Les compositions et les procédés de l'invention peuvent avantageusement être utilisés sur une variété d'échantillons, y compris des cultures de lignées cellulaires et/ou de cellules primaires. Le procédé de préparation est apte à un traitement à haut débit à l'aide de plateformes manuelles ou robotiques.
PCT/US2024/041083 2023-08-08 2024-08-06 Préparation d'échantillon pour acides nucléiques Pending WO2025034736A1 (fr)

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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5210015A (en) 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US6472187B1 (en) * 1999-07-16 2002-10-29 Shimadzu Corporation Method for amplification of RNA
US20030092135A1 (en) * 2001-11-13 2003-05-15 Eppendorf Ag Method and compositions for reversible inhibition of thermostable polymerases
US20030170617A1 (en) 2002-01-28 2003-09-11 Pasloske Brittan L. Crude biological derivatives competent for nucleic acid detection
US20110071031A1 (en) * 2009-09-24 2011-03-24 Qiagen Gaithersburg Inc. Compositions, methods, and kits for isolating and analyzing nucleic acids using an anion exchange material
JP5914338B2 (ja) * 2009-09-24 2016-05-11 キアジェン ゲイサーズバーグ インコーポレイテッド 陰イオン交換材料を使用した核酸の単離および分析のための組成物、方法およびキット
US20170356026A1 (en) * 2011-08-17 2017-12-14 Qiagen Gmbh Methods for RT-PCR comprising an anionic polymer
JP2020156470A (ja) * 2019-03-20 2020-10-01 東洋紡株式会社 非特異増幅が抑制された核酸増幅法
WO2022024935A1 (fr) * 2020-07-29 2022-02-03 東洋紡株式会社 Procédé de limitation de l'amplification non spécifique des acides nucléiques
WO2022024934A1 (fr) * 2020-07-29 2022-02-03 東洋紡株式会社 Procédé amélioré de détection de virus

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5210015A (en) 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
US5487972A (en) 1990-08-06 1996-01-30 Hoffmann-La Roche Inc. Nucleic acid detection by the 5'-3'exonuclease activity of polymerases acting on adjacently hybridized oligonucleotides
US5804375A (en) 1990-08-06 1998-09-08 Roche Molecular Systems, Inc. Reaction mixtures for detection of target nucleic acids
US6214979B1 (en) 1990-08-06 2001-04-10 Roche Molecular Systems Homogeneous assay system
US6472187B1 (en) * 1999-07-16 2002-10-29 Shimadzu Corporation Method for amplification of RNA
US20030092135A1 (en) * 2001-11-13 2003-05-15 Eppendorf Ag Method and compositions for reversible inhibition of thermostable polymerases
US20030170617A1 (en) 2002-01-28 2003-09-11 Pasloske Brittan L. Crude biological derivatives competent for nucleic acid detection
US20110071031A1 (en) * 2009-09-24 2011-03-24 Qiagen Gaithersburg Inc. Compositions, methods, and kits for isolating and analyzing nucleic acids using an anion exchange material
JP5914338B2 (ja) * 2009-09-24 2016-05-11 キアジェン ゲイサーズバーグ インコーポレイテッド 陰イオン交換材料を使用した核酸の単離および分析のための組成物、方法およびキット
US20170356026A1 (en) * 2011-08-17 2017-12-14 Qiagen Gmbh Methods for RT-PCR comprising an anionic polymer
JP2020156470A (ja) * 2019-03-20 2020-10-01 東洋紡株式会社 非特異増幅が抑制された核酸増幅法
WO2022024935A1 (fr) * 2020-07-29 2022-02-03 東洋紡株式会社 Procédé de limitation de l'amplification non spécifique des acides nucléiques
WO2022024934A1 (fr) * 2020-07-29 2022-02-03 東洋紡株式会社 Procédé amélioré de détection de virus

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
"Enzymatic Resource Guide: Polymerases", 1998, PROMEGA
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", May 2005, JOHN WILEY & SONS, INC.
BRESLAUER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 83, 1986, pages 9373 - 9377
DIFFENBACHDVEKSLER: "PCR Primer, A Laboratory Manual", 1995, COLD SPRING HARBOR PRESS
HEID ET AL., GENOME RESEARCH, vol. 6, 1996, pages 986 - 994
HOLLAND ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 7276 - 7280
KWOK ET AL., NUCL. ACID RES., vol. 18, 1990, pages 999 - 1005
LEHNINGERNELSONCOX: "Principles of Biochemistry", 2000, WORTH PUBLISHING
LINJAYSENA, J. MOL. BIOL., vol. 271, 1997, pages 100 - 11
PAVLOV ET AL., TRENDS IN BIOTECHNOL., vol. 22, 2004, pages 253 - 60
TWYMAN: "Advanced Molecular Biology: A Concise Reference", 1999, BIOS SCIENTIFIC PUBLISHERS

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