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WO2016004548A1 - Procédé de pré-traitement de micro-quantités de tissus prélevés par biopsie clinique et enrobés de paraffne - Google Patents

Procédé de pré-traitement de micro-quantités de tissus prélevés par biopsie clinique et enrobés de paraffne Download PDF

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Publication number
WO2016004548A1
WO2016004548A1 PCT/CN2014/000886 CN2014000886W WO2016004548A1 WO 2016004548 A1 WO2016004548 A1 WO 2016004548A1 CN 2014000886 W CN2014000886 W CN 2014000886W WO 2016004548 A1 WO2016004548 A1 WO 2016004548A1
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WIPO (PCT)
Prior art keywords
tissue
genomic dna
slice
paraffin
tumor tissue
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Ceased
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PCT/CN2014/000886
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English (en)
Chinese (zh)
Inventor
崔路漫
赵鑫
侯勇
吴逵
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BGI Genomics Co Ltd
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BGI Genomics Co Ltd
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Priority to PCT/CN2014/000886 priority Critical patent/WO2016004548A1/fr
Priority to CN201480081410.1A priority patent/CN107075559A/zh
Publication of WO2016004548A1 publication Critical patent/WO2016004548A1/fr
Anticipated expiration legal-status Critical
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q

Definitions

  • the invention relates to the field of biotechnology, and relates to a pretreatment method for clinical micro-biopsy paraffin-embedded tissues.
  • pathological biopsy can determine the pathological type and tumor stage by observing the morphological and structural changes of cells and tissues. It is the most accurate and most necessary detection method for tumor diagnosis. Clinically, almost all tumors need to be diagnosed by pathological biopsy. . On this basis, immunohistochemistry, PCR, FISH and other molecular pathological tests to determine the molecular typing of tumors provide an important scientific basis for clinicians to choose appropriate treatment options and prognosis evaluation.
  • PCR polymerase chain reaction
  • FQ-PCR real-time fluorescent quantitative PCR
  • FISH fluorescence in situ hybridization
  • the PCR technology is simple and easy to operate, and the total amount of DNA required is small, but only a single or a small number of genes can be detected at a time; the gene chip can detect a large number of genes at a time, but the false positive rate is high, the accuracy is insufficient, and a special analytical instrument is needed.
  • the first generation sequencing technology was the dideoxynucleotide termination method invented by Sanger et al. in 1977 and the chemical degradation method by Gilbert et al. (Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors [J]. Proc Natl Acad Sci USA, 1977, 74(12): p. 5463-7. Maxam AM, Gilbert WA new method for sequencing DNA [J]. Proc Natl Acad Sci USA, 1977, 74(2): p.560-4.); second generation sequencing technology includes Roche's 454 technology (Margulies, M.
  • 3960-4. is called Third generation sequencing technology.
  • the second-generation sequencing has the advantages of high throughput and high accuracy.
  • the higher sequencing throughput reduces the cost of sequencing and the efficiency of scientific research to a certain extent, and also seeks treatment time for clinical tumor patients.
  • the emergence of high-throughput sequencing technology has revolutionized the molecular testing of clinical laboratories, but how to deal with trace clinical samples has become a major difficulty in clinical applications.
  • the pretreatment method of the paraffin-embedded clinical biopsy tissue provided by the invention can be used for second-generation sequencing, and can also be used for PCR, mass spectrometry, etc., and specifically includes the following steps:
  • the tumor tissue or the suspected tumor tissue is cut out from the tissue slice to be separated according to the HE staining result of the control slice.
  • the tumor tissue or the suspected tumor tissue obtained by the step (4) cutting may be derived from a piece of the tissue slice to be separated, or may be derived from a plurality of slices of the tissue to be separated, generally 2-10 pieces. Specifically, it can be determined according to the needs, as well as the size of the tissue block and the content of the tumor cells. The number of the selected tissue sections to be separated is not necessarily the same, the larger the number of tumor cells in the tissue block, and the smaller the number of tablets, 2 pieces can be .
  • step (1) of the method when the paraffin-embedded clinical biopsy tissue wax block is continuously sliced, the thickness of the slice to be controlled is 4-6 micrometers.
  • the method for performing HE staining may specifically include the following steps: placing the wax at 60 ° C for 30 min; dewaxing by soaking for 10 min in xylene; and thoroughly immersing for 10 min in the new xylene; Dewaxing; immersing in anhydrous ethanol for 5 minutes to remove xylene remaining on the tissue section; immersing in new anhydrous ethanol for 5 minutes to completely remove xylene; immersing in 95% volume fraction of ethanol for 2 minutes to gradually hydrate the section to facilitate nuclear staining; 75% volume fraction of ethanol soaked for 2min; soaked in water for 1min; soaked in new water for 1min; stained for 2min in hematoxylin staining solution for 2min; rinsed with running water for 5min; treated with differentiation solution for 3s; soaked in water for 30s; 75% by volume of ethanol Soaking for 1 min to remove water for alcohol-soluble eosin staining; staining for 10
  • the differentiation liquid comprises anhydrous ethanol, water and HCl.
  • the differentiation liquid is specifically a solution obtained by mixing anhydrous ethanol, water and HCl in a volume ratio of 33: 1.73: 0.35.
  • the tissue slice to be separated may be specifically obtained according to the method comprising the following steps: taking out the HE staining from the serial section of the same clinical biopsy tissue wax block in the step (2) The slice was placed at 60 ° C for 30 min for melting wax; the xylene was immersed for 10 min for dewaxing; the new xylene was immersed for 10 min for complete dewaxing.
  • step (4) of the method the tumor tissue or the suspected tumor tissue is cut from the tissue slice to be separated according to the HE staining result of the control slice, and is performed by a laser capture microdisc.
  • the step (4) is: simultaneously scanning the control slice and the tissue slice to be separated on a stage of a laser capture micro-cutter, and performing HE staining on the control slice. As a result, the position of the tumor tissue or the suspected tumor tissue is determined, and the same position is found in the tissue slice to be separated, and the tumor tissue or the suspected tumor tissue is cut.
  • tumor tissue or suspected tumor tissue obtained from paraffin-embedded clinical biopsy tissue by the method as a nucleic acid extraction sample is also within the scope of the present invention.
  • the application specifically uses the tumor tissue or the suspected tumor tissue as a sample, and the genomic DNA is extracted therefrom by using the nucleic acid automatic extractor.
  • the genomic DNA is extracted from the tumor tissue or the suspected tumor tissue by using the nucleic acid automatic extractor, and specifically, the method includes the following steps:
  • step (a2) adding a lysis buffer to the sample incubated in step (a1), and then transferring the sample all to the reagent cartridge of the nucleic acid automatic extractor, and installing the nucleic acid automatic extractor together with the reagent rack, and The nuclease-free water is added to the elution tube of the nucleic acid automatic extractor, and the procedure is started according to the interface prompting operation of the nucleic acid automatic extractor, and finally the genomic DNA is obtained.
  • Another object of the present invention is to provide a method for genomic second generation sequencing of paraffin-embedded clinical biopsies.
  • the method for performing genome second generation sequencing on paraffin-embedded clinical biopsy tissue may specifically include the following steps:
  • the method for constructing the genomic DNA library to be sequenced by using the genomic DNA may specifically include the following steps:
  • step A the specific parameters used in interrupting the genomic DNA are shown in Table 1.
  • step A the size of the DNA fragment after the fragmentation is mainly 150-300 bp; correspondingly, the size of the DNA fragment in the genomic DNA library to be sequenced obtained in step B is mainly 200-400 bp.
  • step (c) of the method the step of performing quality inspection on the genomic DNA library to be sequenced may be further included before the second generation sequencing of the genomic DNA library to be sequenced.
  • the second generation sequencing may specifically be all types of sequencing methods of Hiseq2000 sequencing (eg, Hiseq PE101+8+101cycle sequencing), Hiseq 2500 or Miseq.
  • the kit for pre-treatment of paraffin-embedded clinical biopsy tissue may specifically include a slicer, a laser capture micro-cutter, an automatic nucleic acid extractor, and a paraffin wax as described above. Instructions for the relevant content of the embedded clinical biopsy tissue pretreatment method.
  • the kit for sequencing second-generation genome of paraffin-embedded clinical biopsy tissue may specifically include a slicer, a laser capture micro-cutter, an automatic nucleic acid extractor, a second-generation sequencer, and the above The instructions for performing the genome second generation sequencing method on the paraffin-embedded clinical biopsy tissue.
  • the microtome is specifically a Finesse 325 manual paraffin slicer manufactured by Thermo Fisher Scientific Co., Ltd.
  • the laser capture microdissection device is specifically a laser capture microdissection device of the Arcturus XT model manufactured by ABI
  • the nucleic acid automatic extractor is specifically a Maxwell MDX model nucleic acid automatic extractor manufactured by Promega Corporation
  • the second generation sequencer is specifically a Ilisea company's Hiseq2000 sequencer.
  • the second-generation sequencer can also be a Hiseq 2500, a Miseq instrument, and the like.
  • Figure 1 shows the results of staining of control sections.
  • Figure 2 shows the tissue sections to be separated before cutting.
  • Figure 3 shows the tissue slice to be separated after cutting.
  • Figure 4 shows the results of the broken product test.
  • Figure 5 shows the library quality test results (2100 detection peak map).
  • Slicer Finenesse 325 manual paraffin slicer from Thermo Fisher Scientific; laser capture microdissection: Arcturus XT laser capture microdisc model from ABI; nucleic acid automatic extractor: Maxwell MDX model from Promega Automatic nucleic acid extractor; second-generation sequencer: Illumina's Hiseq2000 sequencer; DNA interrupter: Covaris product, model E210.
  • the paraffin-embedded clinical biopsy tissue wax block (in which the clinical biopsy tissue tested has been confirmed by clinical diagnosis to contain lung cancer tissue) was serially sliced with a microtome, sliced to a thickness of 4-6 ⁇ m, and placed in warm water at 37 ° C.
  • the tissue sections were unfolded and attached to a specially-labeled special film slide (PNC-FTC-001, name: Polymer Nanowire Chip, brand: FETOLUMINA). Place in a slicing box, store at 4°C for short-term, and store at -80°C for a long time.
  • Baking sheet placed in an oven at 60 ° C for 30 min, melting wax.
  • Neutral gum is sealed to facilitate observation of tissue structure and cell morphology.
  • the formulation of the differentiation solution was as follows: 33 ml of absolute ethanol was taken into the BD tube, and 1.73 ml of water and 0.35 ml of HCl were added to prepare a tube of 35 ml of differentiation liquid.
  • DNA sequencing was performed on conventional paraffin-embedded tissues, and DNA extraction was performed directly after sectioning.
  • the pathological biopsy is through biopsy needles, biopsy forceps to obtain a small piece of tissue, the tumor cell content in the tissue may be only 20%, or even 5%, which will directly affect the sequencing results, reduce the accuracy of genetic diagnosis, and ultimately lead to clinical treatment. failure.
  • the invention adopts an advanced laser capture micro-cutter for tumor tissue separation, and can obtain tumor tissue with tumor cell content of 50%-80%, and obtain higher purity tumor DNA, thereby greatly improving the accuracy of the detection result.
  • the ABI laser capture microdissection instrument was used to separate the tumor tissue. The specific steps are as follows:
  • Fig. 1 The results of the control section staining are shown in Fig. 1.
  • the tissue sections to be separated are shown in Fig. 2 before cutting, and the results after cutting are shown in Fig. 3.
  • Genomic DNA was extracted from the isolated cancer tissue using a Promega automatic extractor. The main steps are as follows:
  • the program ends. According to the interface prompt, the reagent rack is taken out, the DNA in Elution Tubes is collected, and the DNA is detected by Qubit 2.0 fluorescent agent at a concentration of 0.216 ng/ ⁇ L, and the total amount is 19.44 ng.
  • the sample is interrupted as follows:
  • the starting amount of DNA in this example was 19.44 ng, and the library amplification system was strictly operated according to the instructions.
  • the library amplification procedure was operated as follows: 72 ° C for 2 min; 94 ° C for 30 s, 62 ° C for 30 s, 72 ° C for 1 min. , 12 cycles; 72 ° C 5 min; 10 ° C insulation.
  • the number of amplification cycles needs to be changed to 10 cycles.
  • the DNA fragment in the final constructed genomic DNA library was 200-400 bp in length.
  • the object of the present invention is to provide a sample pretreatment method for paraffin-embedded tissues of clinical micro-biopsy, which is beneficial to the requirement of second-generation sequencing of micro-samples, thereby enabling clinicians to better use second-generation sequencing technology for gene detection of tumor samples.
  • the technical scheme adopts laser capture microdissection to perform tumor tissue separation, obtains tissue with higher tumor cell content, and makes the sequencing result more accurate; automated instrument extracts genomic DNA of tissue section, ultra low
  • the initial amount of library construction method, the total amount of DNA as low as 10 ng can also successfully construct a library, using the second generation sequencing technology for genome-wide sequencing.
  • the method of the invention can process samples easily and quickly, obtain more accurate genetic test results, and strive for more time for clinical treatment of tumor patients, and provide a more reliable scientific basis.

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  • Chemical & Material Sciences (AREA)
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Abstract

La présente invention concerne un procédé de pré-traitement de micro-quantités de tissus prélevés par biopsie clinique et enrobés de paraffine. Le procédé comprend les étapes successives suivantes : 1) découpage en tranches d'un bloc de tissu prélevé par biopsie clinique et enrobé de paraffine ; 2) sélection parmi lesdites tranches du même bloc d'une tranche de tissu prélevé par biopsie clinique, et teinture HE de ladite tranche pour obtenir une tranche de référence ; 3) sélection parmi les tranches du même bloc de tissu prélevé par biopsie clinique d'une tranche non teintée HE, et déparaffinage aux fins d'utilisation comme tranche dont le tissu peut être séparé ; 4) sur la base des résultats de teinture HE de la tranche de référence, enlèvement du tissu tumoral par découpage de la tranche dont le tissu peut être séparé.
PCT/CN2014/000886 2014-09-29 2014-09-29 Procédé de pré-traitement de micro-quantités de tissus prélevés par biopsie clinique et enrobés de paraffne Ceased WO2016004548A1 (fr)

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PCT/CN2014/000886 WO2016004548A1 (fr) 2014-09-29 2014-09-29 Procédé de pré-traitement de micro-quantités de tissus prélevés par biopsie clinique et enrobés de paraffne
CN201480081410.1A CN107075559A (zh) 2014-09-29 2014-09-29 临床微量活检石蜡包埋组织的前处理方法

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2018119295A1 (fr) * 2016-12-22 2018-06-28 Ventana Medical Systems, Inc. Procédé entièrement automatisé d'extraction d'acide nucléique destiné à des échantillons de tissu

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Publication number Priority date Publication date Assignee Title
CN108181148A (zh) * 2017-12-25 2018-06-19 天津市海河医院 Ct引导下经皮肺穿刺活检的组织压片及现场评价方法
CN111088216A (zh) * 2019-12-16 2020-05-01 齐妍 一种用于石蜡包埋组织分离检测细胞的方法
CN114689407B (zh) * 2022-04-14 2023-05-19 四川大学华西医院 一种动物微小组织石蜡切片的制作方法

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CN103602726A (zh) * 2013-10-21 2014-02-26 田埂 同时对多种核酸样本进行测序的方法

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CN103602726A (zh) * 2013-10-21 2014-02-26 田埂 同时对多种核酸样本进行测序的方法

Non-Patent Citations (1)

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Title
HUANG, JIAN ET AL.: "Association of Single Nucleotide Polymorphisms of NBS1 Gene With Genetic Susceptibility of Primary Liver Cancer in a Chinese Han Population", PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS, vol. 39, no. 7, 31 December 2012 (2012-12-31), XP055251767 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018119295A1 (fr) * 2016-12-22 2018-06-28 Ventana Medical Systems, Inc. Procédé entièrement automatisé d'extraction d'acide nucléique destiné à des échantillons de tissu
US11390903B2 (en) 2016-12-22 2022-07-19 Ventana Medical Systems, Inc. Fully automated nucleic acid extraction methods for tissue samples

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