A method for extracting nuclei from formaldehyde-fixed and paraffin-embedded samples

Abstract

This invention discloses a method for extracting cell nuclei from formaldehyde-fixed and paraffin-embedded samples, comprising: (1) extracting cell nuclei: cutting PA / PFA-fixed or FFPE samples into pieces, adding cell nuclei extraction solution, incubating, collecting the supernatant, centrifuging, and discarding the supernatant; (2) washing cell nuclei: washing the cell nuclei precipitate from step 1) with PBS mix, centrifuging, discarding the supernatant, and resuspending the cell nuclei with PBS mix. The cell nuclei extracted by this method have advantages such as good morphological integrity, high purity, good RNA integrity, and high RNA yield, which are of great significance for transcriptomics studies of PA / PFA-fixed and FFPE samples.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a method for extracting nuclei from formaldehyde-fixed and paraffin-embedded samples. BACKGROUND

[0002] PA / PFA fixation and FFPE samples are the preferred method for tissue preservation in clinic. However, cross-linking between RNA and other biomolecules of the sample during fixation has a negative impact on the integrity of nucleic acids. Currently, transcriptomic studies of FFPE samples are limited to direct RNA-Seq of the sample, micro-dissection transcriptomics, and spatial transcriptomics. The nuclei of FFPE samples are currently only used for fluorescence in situ hybridization (FISH) and whole genome studies, and have not been applied to transcriptomic studies.

[0003] All the reported FFPE nuclei are extracted at 37℃, but the nuclei extracted at 37℃ are not suitable for subsequent transcriptomic studies. SUMMARY

[0004] The purpose of the present application is to provide a method for extracting nuclei from formaldehyde / polyformaldehyde (PA / PFA) and paraffin-embedded (FFPE) samples.

[0005] TECHNICAL SOLUTION The method for extracting nuclei from formaldehyde-fixed and paraffin-embedded samples of the present application comprises the following steps:

[0006] (1) Extracting nuclei: cut the PA / PFA-fixed or FFPE sample into pieces, add a nuclei extraction solution, incubate, take the supernatant, centrifuge, and discard the supernatant;

[0007] (2) Washing the nuclei: 1) wash the nuclei pellet with a PBS mix solution, centrifuge, discard the supernatant, and resuspend the nuclei with a PBS mix solution.

[0008] Further, the components of the nuclei extraction solution include a surfactant, a protease, and a TE buffer.

[0009] Further, the surfactant includes Sarkosyl, SDS, EDTA-NaCl, or EDTA-KCl, and the protease includes proteinase K, pepsin, trypsin, or collagenase.

[0010] Further, the PBS mix solution has a pH of 7.4 and contains 0.25% RNAase inhibitor.

[0011] The preferred embodiments of the present application are as follows:

[0012] 1) Sample preparation:

[0013] a) Fixed sample: after the biological tissue is dissociated, 4% PA / PFA is added for fixation at 4 DEG C overnight, then cleaned with pre-cooled PBS or SSC buffer solution, cut into 1mm 3 left and right small pieces with a scalpel;

[0014] b) FFPE sample: after the organism is obtained, 4% PA / PFA is added for fixation at 4 DEG C, and after the FFPE sample is prepared, it is stored in a 4 DEG C environment. After the FFPE sample is deparaffinized, 100%, 90%, 70%, 50% ethanol is hydrated, and then a scalpel is used to cut it into 1mm 3 left and right small pieces;

[0015] 2) Extract the cell nucleus: put the cut tissue piece into a 2ml centrifuge tube, add the cell nucleus extraction solution, incubate at 4 DEG C overnight with slight shaking, then take the supernatant to a new centrifuge tube, centrifuge at >10000rpm for 10min, and discard the supernatant;

[0016] 3) Wash the cell nucleus: the cell nucleus precipitate in 2) is washed with PBS mix solution, centrifuged at >10000rpm for 10min, and the supernatant is discarded. Repeat this step 2 times, and then resuspend the cell nucleus with PBS mix solution;

[0017] The cell nucleus extraction solution formula is: surfactant (such as Sarkosyl, SDS, EDTA-NaCl, EDTA-KCl, etc.), protease (such as proteinase K, pepsin, trypsin, collagenase, etc.), TE buffer;

[0018] The composition ratio of the cell nucleus extraction solution is different due to the sample size (such as tissue block, tissue section, etc.) and sample type;

[0019] Beneficial effects: compared with the prior art, the present application has the following advantages: in order to better study the cell nucleus of PA / PFA fixation and FFPE sample from the perspective of transcriptomics, the present application proposes a cell nucleus extraction method for formaldehyde-fixed and paraffin-embedded samples. The method is not only simple to operate, but also has high purity, complete morphology, and high RNA yield and integrity, which meets the later RNA-Seq research, and is expected to meet single cell nucleus sequencing (snRNA-Seq) and even multi-omics research. The present application lays a sample preparation foundation for the transcriptome research of PA / PFA-fixed and FFPE sample cell nucleus, and has important significance. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Flow chart of the cell nucleus extraction method;

[0021] Figure 2 Trypan blue staining bright field microscopic imaging of PFA-fixed mouse brain tissue cell nucleus;

[0022] A, B are the nuclei extracted from the hippocampus of mouse brain tissue (A-10x, B-20x); C is the nuclei extracted from 30um tissue section (20x); D is the nuclei extracted from FFPE sample (mouse brain tissue) (20x);

[0023] Figure 3 A, B are the PI staining microscopic imaging and counting images of the nuclei of the hippocampus of PFA-fixed mouse brain tissue; Figure 4 RNA quality map of PFA-fixed mouse brain tissue and FFPE brain tissue nuclei;

[0024] A is the effect of the extraction temperature of the nuclei on the RNA integrity; B is the effect of the extraction temperature of the nuclei on the RNA yield. DETAILED DESCRIPTION

[0025] Example 1

[0026] 1) Sample preparation: After euthanizing adult mice, their brain tissues were taken, and the hippocampus was taken, and fixed with 4% paraformaldehyde overnight. After fixation, the sample was washed with PBS (1x, pH = 7.4) solution for 3 times, and then cut into 1mm 3 small pieces with a surgical knife.

[0027] 2) Extracting nuclei: Put the cut tissue pieces into a 2ml centrifuge tube, add 1ml of nucleus extraction solution (0.5% Sarkosyl, 1.5mM proteinase K, pH = 8.0 TE buffer, 0.25% RNAase inhibitor), 4°C incubation with slight shaking overnight; after observing the tissue pieces gradually disappearing, transfer the supernatant to a new 1.5ml centrifuge tube, centrifuge at >10000rpm for 10min; wash with PBS mix solution for 3 times, and then resuspend the nuclei with PBS mix;

[0028] 3) Microscopy: After staining the nuclei with trypan blue and PI, microscopic imaging and counting were performed.

[0029] 4) Extracting RNA: Extract the RNA of PFA-fixed brain tissue and nuclei, and detect RIN with Agilent 4150.

[0030] Example 2

[0031] 1) Sample preparation: After euthanizing adult mice, their brain tissues were taken, and the brain tissues were divided into 3-5mm tissue sections, and fixed with 4% paraformaldehyde overnight. After fixation, the sample was washed with PBS (1x, pH = 7.4) solution for 3 times, and then embedded with OCT, and 30um frozen sections were prepared;

[0032] 2) Nucleus extraction: Place the tissue section into a 2ml centrifuge tube, add 1ml of nucleus extraction solution (0.5% Sarkosyl, 0.3mM proteinase K, pH=8.0 TE buffer, 0.25% RNase inhibitor), incubate at 4℃ with gentle shaking for 3h; after observing the tissue block gradually disappear, transfer the supernatant to a new 1.5ml centrifuge tube, centrifuge at >10000rpm for 10min; wash three times with PBS mix solution, and resuspend the nuclei in PBS mix;

[0033] 3) Microscopic examination: After staining the cell nuclei with trypan blue, examine them under a bright-field microscope.

[0034] Example 3

[0035] 1) Sample Preparation: Brain tissue was collected from adult mice after euthanasia. The brain tissue was divided into 3-5 mm sections, fixed overnight with 4% paraformaldehyde, and then stored at 4°C to prepare FFPE samples. The FFPE samples were dewaxed with a dewaxing agent and hydrated with 100%, 90%, 70%, and 50% ethanol before being cut into 1 mm sections with a scalpel. 3 Small pieces on the left and right;

[0036] 2) Extraction of cell nuclei: Place the cut tissue pieces into a 2ml centrifuge tube, add 1ml of cell nucleus extraction solution (0.5% Sarkosyl, 1.5mM proteinase K, pH=8.0 TE buffer, 0.25% RNase inhibitor), incubate at 4°C with gentle shaking overnight; after observing the tissue pieces gradually disappear, transfer the supernatant to a new 1.5ml centrifuge tube, centrifuge at >10000rpm for 10min; wash three times with PBS mix solution, and resuspend the precipitate in PBS mix;

[0037] 3) Microscopic examination: After staining the cell nuclei with trypan blue, the images are examined under a microscope.

[0038] 4) RNA extraction: RNA was extracted from the nuclei of FFPE brain tissue cells and RIN was detected using an Agilent 4150.

Claims

1. A method for extracting cell nuclei from formaldehyde-fixed, paraformaldehyde-fixed, or FFPE-fixed samples, characterized in that, Includes the following steps: (1) Extracting cell nuclei: Cut the formaldehyde-fixed, paraformaldehyde-fixed, or FFPE sample into pieces, add cell nucleus extraction solution, incubate, and after observing the tissue pieces gradually disappear, take the supernatant, centrifuge, and discard the supernatant; The cell nucleus extraction solution is 0.5% Sarkosyl, 1.5mM proteinase K, pH=8.0 TE buffer, and 0.25% RNase inhibitor or 0.5% Sarkosyl, 0.3mM proteinase K, pH=8.0 TE buffer, and 0.25% RNase inhibitor. The incubation conditions were: overnight incubation at 4°C with gentle shaking; (2) Washing the cell nuclei: The cell nucleus precipitate in step (1) was washed with PBS mix solution, centrifuged and the supernatant was discarded. The cell nuclei were then resuspended with PBS mix solution. The PBS mix solution is pH 7.4 PBS with 0.25% RNase inhibitor; If FFPE sample cutting is used in step (1), a dewaxing step with a dewaxing agent is also included before the sample is cut into pieces.

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