WO2016004548A1 - Method for pre-processing micro amounts of paraffin-embedded clinical biopsy tissue - Google Patents

Abstract

Disclosed in the present invention is a method for pre-processing micro amounts of paraffin-embedded clinical biopsy tissue. The method comprises the following steps: 1) successively slicing a block of paraffin-embedded clinical biopsy tissue; 2) selecting a slice from among the slices of the same block of clinical biopsy tissue, and HE staining said slice to obtain a reference slice; 3) selecting a non-HE stained slice from among the slices of said same block of clinical biopsy tissue, and dewaxing so as to use as a slice from which tissue may be separated; 4) on the basis of the HE stain results of the reference slice, cutting tumor tissue off of the slice from which tissue may be separated.

Description

Pretreatment method for clinical micro-biopsy paraffin-embedded tissues Technical field

The invention relates to the field of biotechnology, and relates to a pretreatment method for clinical micro-biopsy paraffin-embedded tissues.

Background technique

In the past 50 years, with the development of the economy and the progress of society, the deterioration of the ecological environment, occupational exposure, unhealthy lifestyles, biological factors and genetic factors, malignant tumors have become a major disease that seriously threatens human health. On February 3, 2014, WHO released the 2014 World Cancer Report. As a populous country, China's new cancer cases and deaths still rank first in the world. According to the latest statistics (Chen Wanqing et al. China's 2010 malignant tumor incidence and death [J]. "China Cancer" 2014 01), China's annual new cancer cases are about 3.12 million, 6 people diagnosed every minute It is a malignant tumor. According to the average life expectancy of 74 years old, the probability of having a malignant tumor in a lifetime is as high as 22%. Problems such as cardiovascular disease, cancer prevention, control and treatment are facing enormous challenges compared to other chronic diseases.

At present, methods for clinical diagnosis of malignant tumors include serological tests, imaging tests such as B-ultrasound, CT, magnetic resonance, endoscopy, and exfoliative cytology, but the only diagnostic gold standard is pathological biopsy. 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.

The rapid development of molecular biology has made genetic diagnosis gradually become a clinical routine laboratory detection technology, widely used in tumor prevention, screening, diagnosis, treatment and prognosis. Commonly used gene diagnostic techniques include polymerase chain reaction (PCR), real-time fluorescent quantitative PCR (FQ-PCR), gene chip, fluorescence in situ hybridization (FISH) and gene sequencing technology. Among them, 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. Equipment, high cost; FISH detection as a common technical means for screening sensitive drugs in breast cancer patients (NNCN.NCCN Clinical Practice Guidelines in Oncology-Breast Cancer Guideline (version 1.2012)), although with good specificity, accurate positioning, etc., but This technique cannot achieve 100% complete hybridization, especially when using shorter cDNA probes. Gene sequencing technology is one of the important methods in modern biological research.

Gene sequencing technology can truly reflect all the DNA genetic information on the genome, and then comprehensively reveal the mechanism and development of tumors, so it plays an important role in the scientific research of tumors. 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. et al. Genome sequencing in microfabricated high-density picolitre reactors [J]. Nature, 2005, 437 (7057): p.376-80.), Illumina M. et al. BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies [J]. Nucleic Acids Res,2006,34(3):p.e22.Turcatti G, et al.A new class of cleavable fluorescent nucleotides:synthesis and optimization as reversible terminators for DNA sequencing by synthesis[J].Nucleic Acids Res,2008,36( 4): p.e25.) and ABI's SOLiD technology; Helicos' single-molecule sequencing technology (Harris TD, et al. Single-molecule DNA sequencing of a viral genome [J]. Science, 2008, 320 (5872) ): p.106-9.Braslavsky I, et al. Sequence information can be obtained from single DNA molecules [J]. Proc Natl Acad Sci USA, 2003, 100(7): p. 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.

As we all know, the sample is the premise of all scientific research, and a large amount of scientific research cannot be carried out because it is difficult to obtain samples. At present, almost all sequencing platforms have high requirements on the quality and total amount of samples, especially the total amount of DNA, which must reach at least the initial amount of 1 μg to meet the experimental processing in the early stage of sequencing. However, a large number of clinical pathological biopsy tissues are only 0.4×0.05×0.05cm. After satisfying the pathological diagnosis, the total amount of remaining DNA is 200-500ng, and only 10-100ng. Such a small amount of DNA samples requires high-throughput whole-genome sequencing, which undoubtedly poses a great challenge to the pre-processing of the sample. Sun Wendong (Xinjiang Institute of Chemistry, Chinese Academy of Sciences. Method for extracting nucleic acids from paraffin-embedded tissues for gene amplification [P]. State Intellectual Property Office of the People's Republic of China: CN 1221794A. July 7, 1999.) A method for extracting nucleic acid from paraffin-embedded tissue for gene amplification is cumbersome and time consuming, and is only suitable for sequencing of a target gene, and is not capable of whole genome sequencing. Han Dianlin et al. (Tiangen Biochemical Technology (Beijing) Co., Ltd.. Method for extracting deoxyribonucleic acid from formalin-fixed paraffin-embedded tissues [P]. State Intellectual Property Office of the People's Republic of China: CN 102146112A. August 10, 2011 Japanese.) A method for extracting DNA from formalin-fixed paraffin-embedded tissues was invented, and the target fragment was obtained by PCR technique. This method also failed to obtain a sufficient amount of genomic DNA for second-generation sequencing. This makes the second-generation sequencing of such advanced technology limited to the processing of large tumor tissue samples for clinical surgical resection, which brings inconvenience to the wide application of clinical. Therefore, how to deal with the trace paraffin-embedded tissue of clinical biopsy is an urgent problem to be solved.

Invention disclosure

It is an object of the present invention to provide a pretreatment method for paraffin-embedded clinical biopsy tissue.

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:

(1) serially sectioning paraffin-embedded clinical biopsy tissue wax blocks;

(2) Taking a slice from the same serial section of the same biopsy tissue wax block for HE staining, The stained section was used as a control section;

(3) taking the un-HE-stained section from the serial section of the same clinical biopsy tissue wax block described in the step (2), and dewaxing it as the tissue slice to be separated;

(4) 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 .

In 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.

In the step (2) of the method, 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 10s in eosin staining solution to stain cytoplasm; immersing in 95% volume fraction of ethanol for 30s to gradually dehydrate tissue sections; soaking 30s in new 95% volume fraction of ethanol Soak for 1min in anhydrous ethanol; soak for 1min in new anhydrous ethanol; soak for 3min in xylene to make the tissue section transparent, easy to observe fine structure , cell morphology; soaked in new xylene for 3min for better transparency; neutral gum seal to facilitate observation of tissue structure, cell morphology.

Wherein, the differentiation liquid comprises anhydrous ethanol, water and HCl.

In one embodiment of the present invention, 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.

In the step (3) of the method, 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.

In 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.

Specifically, 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.

The use of the 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.

In 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.

Specifically, 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:

(a1) adding an incubation buffer and proteinase K to the tumor tissue or the suspected tumor tissue, and incubating at 70 ° C until the liquid is clear and transparent;

(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 provided by the present invention may specifically include the following steps:

(a) pre-treating the paraffin-embedded clinical biopsy tissue to obtain genomic DNA according to the method described above;

(b) constructing a genomic DNA library to be sequenced by the genomic DNA extracted in the step (a);

(c) performing second-generation sequencing of the genomic DNA library to be sequenced obtained in the step (b) to obtain a genomic DNA sequence.

In the step (b) of the method, the method for constructing the genomic DNA library to be sequenced by using the genomic DNA may specifically include the following steps:

A. interrupting the genomic DNA to obtain a DNA fragment after fragmentation;

B. The DNA fragment after the cleavage is sequentially repaired and ligated to obtain a genomic DNA library to be sequenced.

In step A, the specific parameters used in interrupting the genomic DNA are shown in Table 1.

Further, in 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.

In 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.

In step (c) of the method, 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.

It is yet another object of the present invention to provide a kit for pre-treatment of paraffin-embedded clinical biopsy tissue.

The kit for pre-treatment of paraffin-embedded clinical biopsy tissue provided by the present invention 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.

It is yet another object of the present invention to provide a kit for genomic second generation sequencing of paraffin-embedded clinical biopsies.

The kit for sequencing second-generation genome of paraffin-embedded clinical biopsy tissue provided by the present invention 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.

In the present invention, 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. Of course, the second-generation sequencer can also be a Hiseq 2500, a Miseq instrument, and the like.

DRAWINGS

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).

The best way to implement the invention

The following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. For the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.

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.

Example 1. Genomic second generation sequencing of paraffin-embedded clinical biopsies

First, prepare tissue sections

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 μm, 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.

Second, paraffin-embedded tissue sections HE staining:

One slice of the same paraffin tissue serial section was taken for HE staining as a control slice in the next step of tumor tissue isolation. The specific steps are as follows:

1. Baking sheet: placed in an oven at 60 ° C for 30 min, melting wax.

2. Soak for 10 minutes in xylene. The purpose of this step is dewaxing.

3. Soak for 10 minutes in new xylene and dewax thoroughly.

4. Soak for 5 min in absolute ethanol to remove the xylene remaining on the tissue section.

5. Soak for 5 minutes in new anhydrous ethanol to better remove xylene.

6. Immerse for 2 minutes in 95% (volume fraction) ethanol, gradually hydrate the sections and perform nuclear staining.

7, 75% (volume fraction) soaked in ethanol for 2 min.

8. Soak in water for 1 min.

9. Soak in new water for 1 min.

10. Staining in hematoxylin staining solution for 2 min, the cell nucleus was stained in this step.

11. Rinse with running water for 5 minutes.

12. Soak for 3 sec in the differentiation solution.

13. Soak in water for 30 sec.

14, 75% (volume fraction) soaked in ethanol for 1 min, the purpose of this step is to remove water for alcohol-soluble eosin staining.

15. Immerse for 10 sec in the staining of eosin staining solution. This step is cytoplasmic staining.

16. Soak for 30 sec in 95% (volume fraction) ethanol to gradually dehydrate the tissue sections.

17. Soak for 30 sec in a new 95% (volume fraction) ethanol.

18. Soak for 1 min in absolute ethanol.

19. Soak for 1 min in new absolute ethanol.

20, soaked in xylene for 3min, make the tissue section transparent, easy to observe the fine structure, cell morphology.

21, soaked in new xylene for 3min, better transparent.

22. Neutral gum is sealed to facilitate observation of tissue structure and cell morphology.

Among them, 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.

Third, tumor tissue separation

DNA sequencing was performed on conventional paraffin-embedded tissues, and DNA extraction was performed directly after sectioning. However, 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:

1. Prepare sections for tumor tissue separation (control section with HE staining in step 2) From the same paraffin tissue, the baking sheet is carried out according to the steps 1-3 in the second step, and the xylene is dewaxed and taken out to dry to obtain the tissue section to be separated.

2. Place the control section and the tissue section to be separated on the stage of the laser capture microdisc. Scan two slices.

3. Determine the location of the tumor tissue in the control section, find the same position in the tissue section to be separated, cut the tissue, and place it in a 1.5 mL EP tube.

4. 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.

Fourth, extract genomic DNA

Genomic DNA was extracted from the isolated cancer tissue using a Promega automatic extractor. The main steps are as follows:

(1) According to the Promega automatic extractor instructions, add 180 μL of incubation buffer (Maxwell 16 FFPE Plus LEV DNA Purification Kit) and 20 μL of proteinase K to the tumor tissue obtained in step 3, and mark them in a constant temperature dry bath at 70 ° C. Incubate at 900 rpm for 1-24 h. The incubation time is determined by the amount of sample and the change after incubation of the sample. It is best to incubate until the sample becomes clear and transparent.

(2) After the sample is incubated, remove the reagent supplies from the Promega automatic extractor, insert the reagent cartridge and the elution tube (Elution Tubes) into the corresponding positions of the reagent rack, and add 90 μL of enzyme-free water to the Elution Tubes to dissolve. DNA. Since there is a loss in the process of recovering DNA, the smaller the dissolved volume is, the more the total amount is lost. Therefore, the dissolved volume is determined according to the amount of the sample, generally 50-90 μL.

(3) Remove the sample (1) incubated, add 400 μL of Lysis Buffer, transfer all the sample to the reagent cartridge, and install it into the automatic extractor together with the reagent rack, according to the prompt of the extractor interface. Start the program.

(4) After about 30 minutes, 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/μL, and the total amount is 19.44 ng.

5. Interruption and library construction of genomic DNA samples

1. Interruption of genomic DNA samples

Using the genomic DNA obtained in the above step four, the sample is interrupted as follows:

1 According to the quality test results of genomic DNA samples, 10-100 ng of DNA was taken into a 1.5 mL EP tube, and TE buffer was added to make a final volume of 120 μL.

2 Transfer the DNA sample with a final volume of 120 μL into the interrupted tube (Cat. No.: 520052), place it in a specific position where the DNA interrupter interrupts the tube holder, press the instrument green button (DOOR) to open the door, and break the tube holder. Or put the tablet on the rack and pay attention to the right direction! Click "Configure" and set the parameters as shown in Table 1:

Table 1 genomic DNA breaking parameters

Interrupt the fragments that are mainly 150-300 bp.

3 Slowly aspirate the interrupted sample and transfer it to a 1.5 mL non-stick tube. Purify the magnetic beads provided by the Ovation Ultralow Library System and add 2 volumes of magnetic beads to the sample for purification (see the kit instructions for specific procedures).

4 Run the DNA fragment obtained in the previous step on the Agilent 2100 Bioanalyzer High Sensitivity DNA Assay. The results of the interrupted product test are shown in Figure 4 (except for the marker). It can be seen from the figure that the interrupted product fragments are mainly distributed at 150-300 bp, and the main peak is at 194 bp, which meets the requirements for database construction.

2. Construction of genomic DNA library

Application Ovation Ultralow Library System (product of NUGEN, catalog number 0303).

1 End repair: Follow the instructions.

2 Connector: Follow the instructions.

3 ligation product purification: follow the instructions.

4 Library amplification: 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.

When the initial amount of DNA is 10 ng, the number of amplification cycles needs to be changed to 14 cycles;

When the initial amount of DNA is 30 ng, the number of amplification cycles needs to be changed to 13 cycles;

When the starting amount of DNA is higher than 40 ng, the number of amplification cycles needs to be changed to 10 cycles.

5 Purification of the amplified library: follow the instructions.

The DNA fragment in the final constructed genomic DNA library was 200-400 bp in length.

Sixth, the library quality inspection

(1) The library quality was detected by Agilent 2100 Bioanalyzer and real-time PCR (QPCR). The 2100 detection peak is shown in Figure 5. The results are shown in Table 2:

Table 2 Agilent 2100 Bioanalyzer and real-time PCR results

The results indicated that most of the fragments of the library were concentrated at 286 bp, and the concentrations were consistent with the sequencing requirements of the machine. Hiseq 2000 sequencing on the machine sequencing QPCR molar concentration is greater than 5nM, less than 500nM, the fragment distribution between 200-400bp meets the requirements of the machine.

(2) The rest of the library quality control requirements are the same as those required for the Illumina library.

Seven, Hiseq2000 sequencing

Based on the concentration measured by QPCR in Table 2 of step 6 (1), the above qualified library was sequenced by Hiseq PE101+8+101cycle. The specific operation procedure is detailed in the Hiseq operating instructions.

Eight, sequencing results comparison

Using Hg19 as the human genome reference sequence, the sequencing results were compared with the reference sequence, and the alignment rate was 99.49%, and the coverage was 98.51%, which proved that the whole genome sequence was measured, and the sample of this example was successfully processed.

Industrial application

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. . Compared with other existing technologies, 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.

Claims (16)

A pretreatment method for paraffin-embedded clinical biopsy tissue, comprising the following steps: (1) serially sectioning paraffin-embedded clinical biopsy tissue wax blocks; (2) taking one slice of the same piece of clinical biopsy tissue wax block, performing HE staining, and the stained slice is used as a control slice; (3) taking the un-HE-stained section from the serial section of the same clinical biopsy tissue wax block described in the step (2), and dewaxing it as the tissue slice to be separated; (4) 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 method according to claim 1, wherein in the step (1), when the paraffin-embedded clinical biopsy tissue wax block is continuously sliced, the thickness of the control slice is 4-6 μm. The method according to claim 1 or 2, wherein in the step (2), the method for performing HE dyeing comprises the steps of: placing at 60 ° C for 30 min; soaking in xylene for 10 min; soaking in new xylene 10 min; soak for 5 min in absolute ethanol; soak for 5 min in new absolute ethanol; soak for 2 min in 95% volume fraction of ethanol; soak for 2 min in 75% volume fraction of ethanol; soak for 1 min in water; soak for 1 min in new water; hematoxylin staining Staining in liquid for 2 min; washing in running water for 5 min; treatment in differentiation solution for 3 s; soaking in water for 30 s; soaking in 75% by volume of ethanol for 1 min; dyeing in eosin staining solution for 10 s; soaking in 95% by volume of ethanol for 30 s; new 95% Soak in volume fraction of ethanol for 30s; soak for 1min in anhydrous ethanol; soak for 1min in new anhydrous ethanol; soak for 3min in xylene; soak for 3min in new xylene; seal with neutral gum. The method of claim 3 wherein said differentiation fluid comprises anhydrous ethanol, water and HCl. The method according to any one of claims 1 to 4, wherein in the step (3), the tissue slice to be separated is obtained according to a method comprising the following steps: the same example as described in the step (2) Sections without HE staining were taken from serial sections of clinical biopsy tissue wax blocks; placed at 60 ° C for 30 min; soaked in xylene for 10 min; soaked in new xylene for 10 min. The method according to any one of claims 1 to 5, wherein in step (4), 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. Down, it was carried out using a laser capture microdisc. The method according to claim 6, wherein 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, In the control section, the position of the tumor tissue or the suspected tumor tissue is determined according to the result of HE staining, and the same position is found in the tissue section to be separated, and the tumor tissue or the suspected tumor tissue is cut. Use of the tumor tissue or suspected tumor tissue obtained from paraffin-embedded clinical biopsy tissue by a method according to any one of claims 1-7 as a nucleic acid extraction sample. The use according to claim 8, wherein the application is to extract genomic DNA from the tumor tissue or the suspected tumor tissue using the nucleic acid automatic extractor. The use according to claim 9, characterized in that the extraction of genomic DNA from the tumor tissue or the suspected tumor tissue using the nucleic acid automatic extractor is carried out according to the method comprising the following steps: (a1) adding an incubation buffer and proteinase K to the tumor tissue or the suspected tumor tissue, and incubating at 70 ° C until the liquid is clear and transparent; (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. A method for sequencing genomic second generation of paraffin-embedded clinical biopsy tissue, comprising the following steps: (a) pre-treating a paraffin-embedded clinical biopsy tissue to obtain genomic DNA according to the method of any of claims 1-10; (b) constructing a genomic DNA library to be sequenced by the genomic DNA extracted in the step (a); (c) performing second-generation sequencing of the genomic DNA library to be sequenced obtained in the step (b) to obtain a genomic DNA sequence. The method according to claim 11, wherein in the step (b), the method for constructing the genomic DNA library to be sequenced by using the genomic DNA comprises the following steps: A. interrupting the genomic DNA to obtain a DNA fragment after fragmentation; B. The DNA fragment after the cleavage is sequentially repaired and ligated to obtain a genomic DNA library to be sequenced. The method according to claim 11 or 12, wherein in step (c), the second step of sequencing the genomic DNA library to be sequenced further comprises the step of performing a quality check on the genomic DNA library to be sequenced. The method according to any one of claims 11-13, wherein in the step (c), the second generation sequencing is Hiseq2000 sequencing, Hiseq2500 sequencing or Miseq sequencing. A kit of pre-treated paraffin-embedded clinical biopsy tissue, including a microtome, a laser capture microdisc, and instructions incorporating the method of any of claims 1-10. A kit for sequencing second-generation genomes of paraffin-embedded clinical biopsy tissue, including a microtome, a laser capture microscopy instrument, an automated nucleic acid extractor, a second generation sequencer, and recited in claims 11-14 Instructions for any of the methods described.

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