CN110835628A - Paraffin removal lysate for extracting genome DNA of paraffin section, extraction kit and extraction method - Google Patents
Paraffin removal lysate for extracting genome DNA of paraffin section, extraction kit and extraction method Download PDFInfo
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Abstract
The application relates to a nucleic acid extraction method, in particular to paraffin removal lysate for extracting genome DNA of paraffin sections, an extraction kit and an extraction method. The kit comprises paraffin removal lysate, binding buffer solution, washing solution I, washing solution II and eluent, wherein a paraffin removal agent in the paraffin removal lysate adopts 1, 3-propane diamine. The dewaxing lysate adopts 1, 3-propane diamine as a dewaxing agent to be directly added into the dewaxing lysate, dewaxing and cracking are carried out synchronously, after cracking and dewaxing are finished, additional operation is not needed, combination, washing and elution can be directly carried out, the dewaxing lysate is very suitable for the existing popular nucleic acid extraction and purification instrument, and any operation is not needed to be carried out in the midway by manpower as long as a sample and a corresponding reagent are put in.
Description
Technical Field
The application relates to a nucleic acid extraction method, in particular to paraffin removal lysate for extracting genome DNA of paraffin sections, an extraction kit and an extraction method.
Background
Paraffin section (paraffinsection) is the most widely used method in routine histological slide-making techniques. Paraffin sections are not only used for observing the morphological structure of normal cell tissues, but also are the main methods used by subjects such as pathology and forensic science to study, observe and judge morphological changes of cell tissues, and have been widely used in the study of other subjects. Since fresh specimens are difficult to obtain, retrospective studies of existing cases can only be performed by extracting DNA from paraffin-embedded tissues, and thus paraffin-embedded tissues become the most valuable resource from which to source.
The Chinese invention patent application (publication number: CN104164418A, published: 2014.06.26) discloses a kit for extracting genome DNA from a paraffin-embedded tissue section and a using method thereof, wherein the kit comprises a dewaxing agent and 0.9-1.1 parts of xylene; the dewaxing cleaning agent consists of 1.8-2.2 parts by volume of 95% ethanol and 0.9-1.1 part by volume of 75% ethanol; 0.28-0.32 volume part of digestive juice, wherein the digestive juice is prepared by SDS, Tris-HCl, EDTA, proteinase K and water, and the concentration of each component in the digestive juice is 0.9-1.1% of SDS, 0.9-1.1mM of Tris-HCl 9-11 mM of EDTA and 90-110 mu g/ml of proteinase K; the protein precipitator is 4MNH4AC solution DNA precipitator, and the DNA precipitator is independently packaged 3M sodium acetate solution, absolute ethyl alcohol and 70% ethyl alcohol. The method adopts a DNA precipitation method, adopts dimethylbenzene as a dewaxing agent, and uses dimethylbenzene as a toxic substance, so that the method is toxic to test operators and is not beneficial to environmental protection and discharge.
The Chinese patent application (publication number: CN108998445A, published: 2018.12.14) discloses a kit for extracting nucleic acid from a paraffin section sample, which comprises dewaxed oil, wherein the dewaxed oil comprises n-hexadecane and turpentine, and the volume ratio of the n-hexadecane to the turpentine is (3-6): 1. the dewaxing oil is used for dewaxing paraffin slice samples, toxic chemicals are not needed, and the n-hexadecane and the turpentine oil are matched for use, so that the dewaxing oil has a good dewaxing effect, is non-toxic and environment-friendly, and simplifies complex washing steps in a dewaxing process. In the method, after cracking is finished, layering is carried out, dewaxing cracking liquid needs to be manually sucked, if the dewaxing layer is arranged on the upper layer, the sucked gun head penetrates through an organic matter-wax layer, certain requirements on the suction requirement can be met, the gun head can possibly bring the organic matter-wax layer out, and if the lower layer dewaxing cracking liquid is sucked too much or too little, subsequent extraction can be influenced.
The existing DNA extraction methods such as phenol chloroform method, salting-out method, adsorption column method, immunoaffinity method, magnetic bead method and the like can be mainly used for extracting saliva DNA, but the adsorption column method has obvious defects in extraction effect and extracted DNA concentration, and the magnetic bead method has many points to be improved in simple operation and extraction effect.
Disclosure of Invention
In order to solve the technical problem that the lower part operation is required to be carried out after the dewaxing and cracking, the application aims to provide the dewaxing lysate for extracting the genome DNA of the paraffin section, the dewaxing lysate adopts 1, 3-propane diamine as a dewaxing agent to be directly added into the dewaxing lysate, dewaxing and cracking are carried out synchronously, after the cracking and dewaxing are finished, additional operation is not required, the dewaxing solution can be directly combined, washed and eluted, the dewaxing solution is very suitable for the existing popular nucleic acid extraction and purification instrument, and any operation is not required to be carried out in the midway by manpower as long as a sample and a corresponding reagent are put in the nucleic acid extraction and purification instrument.
In order to achieve the above object, the present application adopts the following technical solutions:
the paraffin removal lysate for extracting the genome DNA of the paraffin section comprises the following components:
the above-mentioned percentage is mass percentage, the rest is water, pH is 8.5-9.0.
Preferably, the dewaxing lysate is composed of the following components:
the above-mentioned percentage is mass percentage, the rest is water, pH is 8.5-9.0.
In addition, the application also discloses an extraction kit of the paraffin section genome DNA, which comprises paraffin removal lysate, binding buffer solution, washing solution I, washing solution II and eluent, wherein the paraffin removal lysate adopts the paraffin removal lysate.
Preferably, the binding buffer solution is isopropanol with the mass percent of 75-85% and the pH value is 7.0-8.0.
Preferably, the washing solution I comprises 2.5-3.5MGuHCl, 8-12mM Tris, 45-55% ethanol, pH 7.5-8.5; washing solution II comprises 8-12mM Tris, 75-85% ethanol, pH 7.5-8.5.
Preferably, the eluent comprises: 8-12mM Tris, 0.05-0.15mM EDTA; pH8.5-9.5.
Preferably, the kit also comprises proteinase K solution, and the proteinase K15-22 mg/ml. The applicant has found that addition of proteinase k at the appropriate time facilitates lysis of the sample.
Preferably, the kit comprises the following components:
1) wax removal cracking liquid: 30%1, 3-propanediamine, 0.1M Tris-HCl, 20M EDTA, 1.2M NaCl, 4M CuSCN, 1% SDS, 2% PVP, pH 8.9;
2) binding buffer: 80% isopropyl alcohol; pH7.5;
3) washing solution I: 3MGuHCl, 10mMTris, 50% ethanol; pH8.0;
4) washing solution II: 10mM Tris, 80% ethanol; pH7.5;
5) eluent: 10mM Tris, 0.1mM EDTA; pH9.0;
6) proteinase k.
The application also discloses a method for slicing the genome DNA by paraffin, and the method adopts the kit. The method comprises the following steps:
1) taking a tissue paraffin section, transferring the tissue paraffin section to an EP tube, adding a paraffin removal lysis solution, and uniformly mixing by shaking;
2) adding protease k, heating and shaking at 60-70 deg.C for 20-40min, standing in 75-85 deg.C water bath for 15-25min, cooling to room temperature, supplementing protease k, heating and shaking at 60-70 deg.C for 5-15 min;
3) adding binding buffer solution and magnetic beads, and rotating on a vertical mixer for 10-20 min;
4) placing the centrifuge tube on a magnetic frame for 0.5-2.0min until the magnetic beads in the tube are completely adsorbed, and removing all liquid;
5) taking the centrifugal tube out of the magnetic frame, adding a washing solution I, and carrying out vortex oscillation on the centrifugal tube for 5-10 times to fully disperse magnetic beads; 6) then placing the centrifugal tube on a magnetic separation frame for 0.5-2.0min until the magnetic beads in the tube are completely adsorbed, and removing all supernatant;
6) adding a washing solution II, and performing vortex oscillation on the centrifugal tube for 5-10 times to fully disperse the magnetic beads; then placing the centrifugal tube on a magnetic separation frame for 0.5-2.0min until the magnetic beads in the tube are completely adsorbed; removing all supernatant;
7) repeating the step 6);
8) adding eluent, shaking at 60-70 deg.C for 4-8min, placing the centrifuge tube in a magnetic separation rack for 1.5-2.5min, and transferring the supernatant to another clean centrifuge tube with a gun head; store at-20 ℃.
Preferably, the adding amount of the proteinase k for the first time is 3-5% of the volume of the dewaxing lysis solution, and the added proteinase k is 3-5% of the volume of the dewaxing lysis solution.
Due to the adoption of the technical scheme, the method has the following characteristics:
1) the dewaxing/cracking is synchronously carried out, the individual dewaxing is not needed, the dewaxing/cracking can be directly used for extracting the genome DNA after cracking, the time and the labor are saved, and the efficient and complete dewaxing effect can be achieved through the 1, 3-propane diamine dewaxing agent;
2) the reagents cannot influence each other, dewaxing and cracking are carried out synchronously, only 1 hour is needed for dewaxing and cracking, the dewaxed tissue can be fully cracked, independent dewaxing and centrifugation are not needed, time and labor are saved, and the dewaxed tissue can be directly used for extracting genome DNA after cracking;
3) the method is suitable for manual extraction, can be matched with a nucleic acid extractor in a microplate in an automatic mode, and has high extraction purity and simple, convenient and safe operation;
4) the method is suitable for embedding paraffin sections of different tissue types;
5) high concentration, purity: combined with magnetic beads, the extracted DNA has high concentration and purity, and can be directly used for downstream experiments.
6) The kit has good stability.
7) The reagent does not contain toxic solvents such as phenol, chloroform and the like.
Drawings
FIG. 1 is an electrophoretogram of paraffin sections of different tissues.
FIG. 2 is a diagram showing PCR program setup.
FIG. 3 is a DNAPCR electrophoresis image of paraffin sections of different tissues.
Detailed Description
Primary reagents and instruments
The instrument comprises the following steps: the centrifuge, the water bath, the constant temperature mixer, the vortex oscillation instrument, the vertical mixer, the 2mLEP pipe and the magnetic frame adopt conventional experimental equipment.
Reagent: 1, 3-propanediamine, proteinase k, Tris, EDTA, NaCl, GuSCN, SDS, PVP, GuHCl are produced by sigma;
ethanol and isopropanol are produced by the national medicine group;
magnetic beads were produced by Qiagen (from MagattractDNAkit).
Extraction formula
The kit comprises the following components:
1) wax removal cracking liquid: 0.1M Tris-HCl, 20M EDTA, 1.2M NaCl, 4M GluSCN, 1% SDS, 2% PVP, pH 8.9;
2) wax removing agent: 1, 3-propanediamine;
3) binding buffer: 80% isopropyl alcohol; pH7.5;
4) washing solution I: 3MGuHCl, 10mMTris, 50% ethanol; pH8.0;
5) washing solution II: 10mM Tris, 80% ethanol; pH7.5;
6) eluent: 10mM Tris, 0.1mM EDTA; pH9.0;
7) proteinase k20 mg/ml.
Extraction step
1) Taking 2-8 FFPE tissue slices, transferring the FFPE tissue slices to a 2mLEP tube, adding 600 mu L of paraffin removal and lysis solution, and shaking and uniformly mixing.
2) Adding 20 μ L proteinase k, heating at 65 deg.C and shaking for 30min, standing in 80 deg.C water bath for 20min, cooling to room temperature, supplementing 20 μ L proteinase k, and heating at 65 deg.C and shaking for 10 min.
3) Add 300. mu.LBindingBuffer and 10. mu.LMagnet Beads and place on a vertical mixer and rotate for 15 min.
4) And (4) putting the centrifugal tube on a magnetic frame for 1min until the magnetic beads in the tube are completely adsorbed. The tip is used to remove as much liquid as possible without contacting the beads.
5) And taking the centrifugal tube out of the magnetic frame, adding 500 mu of LWashBufferI, and vortexing the centrifugal tube for 5-10 times to fully disperse the magnetic beads. Then the centrifuge tube was placed on a magnetic separation rack for 1min until the beads in the tube were completely adsorbed. The whole supernatant was removed.
6) Adding 500 mu of LWashBufferII, and vortexing the centrifuge tube for 5-10 times to fully disperse the magnetic beads. Then the centrifuge tube was placed on a magnetic separation rack for 1min until the beads in the tube were completely adsorbed. The whole supernatant was removed.
7) Repeat step 6).
8) 50 μ LElutionbuffer was added and shaken at 65 ℃ for 5 min. The centrifuge tube was then placed on a magnetic separation rack for 2min and the supernatant was transferred to another clean centrifuge tube with a pipette tip.
9) Paraffin section tissue genomic DNA was stored at-20 ℃.
Test example 1
Agarose gel electrophoresis detection
1. Paraffin sections embedded in different tissues were extracted with the present application followed by agarose gel electrophoresis.
2. Preparing glue: 0.05g of agarose was weighed, 5ml of 1XTAE Buffer was added, shaken well and then placed in a microwave oven to be heated to boiling. Taking out, standing, cooling, adding 3ul of coloring agent, and shaking uniformly. Pour into electrophoresis plate and wait for gel.
3. Dispensing: 5ul loading Buffer and 10ul DNA were mixed and spotted into the gel. And opening the electrophoresis apparatus and starting to run the gel.
A map of the electrophoresis of paraffin sections from 4 different tissues is shown in figure 1.
Paraffin section genome DNA PCR
PCR sample application System
PCR program set-up As shown in FIG. 2, FIG. 3 is a PCR electrophoretogram of genomic DNA from different paraffin sections. And (4) conclusion: the extracted paraffin section has higher concentration of genome DNA and larger fragment.
Test example 2
5 parts of different paraffin tissues are taken, 10 slices of each paraffin tissue are continuously sliced, and 5 slices are taken according to each group. The kit provided by the embodiment of the application is used for extracting nucleic acid from the first group, a group of comparison examples are set in a comparison mode, xylene is used for replacing 1, 3-propane diamine in the comparison examples to extract nucleic acid from the second group of samples, and the method provided by the application is used for extracting nucleic acid from both groups.
The concentration and purity of the extracted nucleic acid were measured by a nucleic acid microanalyzer (Agilent 2100 Biochemical Analyzer), and the measured concentrations and purities are shown in tables 1 and 2.
Table 1 shows the results of concentration of a group of nucleic acid samples
Table 2 shows the results of concentration of two sets of nucleic acid samples
As can be seen from tables 1 and 2, the concentration of the nucleic acid extracted from the first group is significantly better than that of the second group, but the purity is not much different, but the dewaxing effect of the sample by using the 1, 3-propane diamine in the present application is better than that of xylene, so that the 1, 3-propane diamine in the present application can replace the xylene conventionally used for dewaxing paraffin samples.
Test example 3
In this test example, 10 groups of formalin-fixed paraffin-embedded liver tissue samples of sick mice were used, and the storage time of 10 groups of sample tissues from the start of fixed embedding to the time of experiment was 1 month, 2 months, 3 months, 4 months, 8 months, 10 months, 12 months, 2 years, 3 years, 4 years, and 5 years, respectively. Wherein 5 specimens in 1 year are numbered as 1-5; 5 specimens of 2-5 years are numbered as 6-10 in sequence.
The method provided by the present application was used to extract nucleic acids from the 12 sets of samples (10 serial sections each), and then the concentration and purity of the 12 sets of nucleic acids were measured using a nucleic acid micro-meter (agilent 2100 biochemical analyzer), and the measured concentrations and purities are shown in table 3 below.
Table 3 concentration results for samples
As can be seen from table 3, the kit provided in the present application can extract DNA from paraffin section samples over 5 years.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present disclosure, including any person skilled in the art, having the benefit of the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The paraffin removal lysate for extracting the genome DNA of the paraffin section is characterized by comprising the following components:
20.0-40.0% of 1, 3-propane diamine;
Tris-HCl 0.05-0.15M；
EDTA 15.0-25.0mM；
NaCl 1.0-1.5M；
GuSCN 3.0-5.0M；
SDS 0.05-0.15%；
PVP 1.50-2.50%；
the above-mentioned percentage is mass percentage, the rest is water, pH is 8.5-9.0.
2. The paraffin-removed lysate for extracting genomic DNA from paraffin sections according to claim 1, which is composed of the following components:
25.0-35.0% of 1, 3-propane diamine;
Tris-HCl 0.08-0.12M；
EDTA 18.0-22.0mM；
NaCl 1.0-1.5M；
GuSCN 3.5-4.5M；
SDS 0.05-0.15%；
PVP 1.50-2.50%；
the above-mentioned percentage is mass percentage, the rest is water, pH is 8.5-9.0.
3. An extraction kit of paraffin section genome DNA, which comprises paraffin removal lysate, binding buffer solution, washing solution I, washing solution II and eluent, and is characterized in that the paraffin removal lysate adopts the paraffin removal lysate of claim 1 or 2.
4. The kit for extracting genomic DNA from paraffin sections according to claim 1, wherein the binding buffer is isopropanol 75-85% by mass, pH 7.0-8.0.
5. The kit for extracting genomic DNA from paraffin sections according to claim 1, wherein the washing solution I comprises 2.5-3.5MGuHCl, 8-12mM Tris, 45-55% ethanol, pH 7.5-8.5; washing solution II comprises 8-12mM Tris, 75-85% ethanol, pH 7.5-8.5.
6. The kit for extracting genomic DNA from paraffin sections according to claim 1, wherein the eluent comprises: 8-12mM Tris, 0.05-0.15mM EDTA; pH8.5-9.5.
7. The kit for extracting genomic DNA from paraffin sections according to claim 1, wherein the kit further comprises proteinase K solution, proteinase K15-22 mg/ml.
8. The kit for extracting genomic DNA from paraffin sections according to claim 1, wherein the kit comprises the following components:
1) wax removal cracking liquid: 30%1, 3-propanediamine, 0.1M Tris-HCl, 20M EDTA, 1.2M NaCl, 4M CuSCN, 1% SDS, 2% PVP, pH 8.9;
2) binding buffer: 80% isopropyl alcohol; pH7.5;
3) washing solution I: 3MGuHCl, 10mMTris, 50% ethanol; pH8.0;
4) washing solution II: 10mM Tris, 80% ethanol; pH7.5;
5) eluent: 10mM Tris, 0.1mM EDTA; pH9.0;
6) proteinase k.
9. A method for paraffin sectioning of genomic DNA, which comprises using the kit according to any one of claims 1 to 7; the method comprises the following steps:
1) taking a tissue paraffin section, transferring the tissue paraffin section to an EP tube, adding a paraffin removal lysis solution, and uniformly mixing by shaking;
2) adding protease k, heating and shaking at 60-70 deg.C for 20-40min, standing in 75-85 deg.C water bath for 15-25min, cooling to room temperature, supplementing protease k, heating and shaking at 60-70 deg.C for 5-15 min;
3) adding binding buffer solution and magnetic beads, and rotating on a vertical mixer for 10-20 min;
4) placing the centrifuge tube on a magnetic frame for 0.5-2.0min until the magnetic beads in the tube are completely adsorbed, and removing all liquid;
5) taking the centrifugal tube out of the magnetic frame, adding a washing solution I, and carrying out vortex oscillation on the centrifugal tube for 5-10 times to fully disperse magnetic beads; 6) then placing the centrifugal tube on a magnetic separation frame for 0.5-2.0min until the magnetic beads in the tube are completely adsorbed, and removing all supernatant;
6) adding a washing solution II, and performing vortex oscillation on the centrifugal tube for 5-10 times to fully disperse the magnetic beads; then placing the centrifugal tube on a magnetic separation frame for 0.5-2.0min until the magnetic beads in the tube are completely adsorbed; removing all supernatant;
7) repeating the step 6);
8) adding eluent, shaking at 60-70 deg.C for 4-8min, placing the centrifuge tube in a magnetic separation rack for 1.5-2.5min, and transferring the supernatant to another clean centrifuge tube with a gun head; store at-20 ℃.
10. The method for extracting genomic DNA from paraffin sections according to claim 9, wherein the first addition of proteinase k is 3-5% of the volume of the paraffin-removed lysate, and the additional proteinase k is 3-5% of the volume of the paraffin-removed lysate.
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| CN113755486A (en) * | 2021-09-23 | 2021-12-07 | 翌圣生物科技(上海)股份有限公司 | Paraffin remover for processing paraffin-embedded samples and method for extracting nucleic acid from paraffin-embedded samples |
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| PALAK G. PATEL ET AL.: "Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction", 《JOURNAL OF VISUALIZED EXPERIMENTS》 * |
| ZSOLT KOVACS ET AL.: "DNA extraction from paraffin embedded colorectal carcinoma samples: A comparison study of manual vs automated methods,using four commercially kits", 《WORLD JOURNAL OF CLINICAL ONCOLOGY》 * |
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| CN113308460A (en) * | 2020-12-15 | 2021-08-27 | 深圳未知君生物科技有限公司 | Kit for extracting bacterial DNA in paraffin section |
| CN113088515A (en) * | 2021-05-07 | 2021-07-09 | 杭州康代思锐生物科技有限公司 | Kit and method for extracting FFPE tissue sample DNA and application thereof |
| CN113755486A (en) * | 2021-09-23 | 2021-12-07 | 翌圣生物科技(上海)股份有限公司 | Paraffin remover for processing paraffin-embedded samples and method for extracting nucleic acid from paraffin-embedded samples |
| CN115011590A (en) * | 2022-06-28 | 2022-09-06 | 中国科学院生态环境研究中心 | Method for extracting DNA inside and outside environmental microorganism cells by using paramagnetic particle method |
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| CN110835628B (en) | 2021-07-27 |
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