CN113322303A

CN113322303A - Nucleic acid extracting solution for paraffin section sample and extracting method

Info

Publication number: CN113322303A
Application number: CN202110513982.4A
Authority: CN
Inventors: 宋莲; 翁国武; 季昱
Original assignee: Nanjing Zhongkebio Medical Technology Co ltd
Current assignee: Nanjing Zhongkebio Medical Technology Co ltd
Priority date: 2021-05-12
Filing date: 2021-05-12
Publication date: 2021-08-31

Abstract

The invention discloses a nucleic acid extracting solution for a paraffin section sample, which comprises the following components: 5 to 30 percent of liquid dewaxing agent, 10 to 300mM of Tris-HCl, 1 to 100mM of EDTA, 0.02 to 3 percent of LDS, 0.05 to 3M of guanidine hydrochloride, 0.05 to 5 percent of N-acetylcysteine, 1 to 30mg/ml of potassium chloride, 0.05 to 1M of sodium chloride, 0.02 to 2M of sodium hydroxide, 0.1 to 10 percent of triton and 0.1 to 5 percent of Tween 20. The nucleic acid extracting solution provided by the invention can provide a strong alkaline environment to promote the release of cell membrane broken nucleic acid, meanwhile, LDS can effectively remove impurities such as protein and the like, N-acetylcysteine can reduce the viscosity of a sample and assist the cell membrane breakage, the whole extraction process does not need complex operations such as dewaxing, lysate, washing, elution and the like, the complex operations such as a centrifuge, an extractor and the like are not needed, the operation is simple and convenient, the efficiency is high, and the extraction cost is reduced.

Description

Nucleic acid extracting solution for paraffin section sample and extracting method

Technical Field

The invention belongs to the technical field of nucleic acid extraction, and particularly relates to a nucleic acid extracting solution for a paraffin section sample and an extraction method.

Background

Formalin-fixed paraffin-embedded tissue is often used for retrospective study of large numbers of cases as one of the most convenient sources of clinical material. In recent years, with the emergence of many molecular markers related to disease diagnosis, prognosis and treatment, gene detection and screening at molecular level using paraffin tissue sections as materials has become a routine means for clinical diagnosis and related research. For various genetic analysis methods, it is important to first obtain nucleic acids of sufficient purity and quantity.

Most of the existing methods and kits for extracting nucleic acid from paraffin section samples are mainly operated manually, and the whole operation flow is relatively complicated and takes more than 4 hours; a small part of the liquid is extracted by an automatic instrument containing a mechanical arm for transferring liquid, the instrument belongs to a large-scale automatic workstation, the instrument cost is high, and the instrument is not suitable for extracting a small amount of samples; the existing extraction method which is used more in the market mainly adopts a centrifugal column extraction method and a miniaturized nucleic acid extractor extraction method; centrifugal column and miniaturized nucleic acid extraction instrument extraction methods, most of which need to be processed through steps of dewaxing, lysate, combination, washing, elution and the like; the centrifugal column extraction method needs to use a centrifugal machine for centrifugation for multiple times, has a complex process and cannot realize automatic extraction; the extraction characteristic of the small nucleic acid extractor is that the magnetic beads combined with nucleic acid are transferred to buffer solution systems of washing, elution and the like by moving a magnetic rod, and finally the purpose of nucleic acid extraction and purification is realized, but the small automatic machine has small flux, a nucleic acid extraction method needing to add reagents step by step can not be applied to the machine to realize full automation, and semi-automatic extraction needs to be completed by combining manual operation, although some extraction methods realize the full-automatic extraction combining dewaxing and cracking liquid with one step, the method still has certain problems, such as poor extraction effect caused by insufficient cracking liquid, insufficient dewaxing and the like, and more magnetic beads remain in nucleic acid hole sites, thereby causing downstream detection failure, so that most manufacturers adopt the semi-automatic extraction combining the manual machine for dewaxing and cracking steps in the process of extracting nucleic acid from paraffin slice samples, the operation process is complicated and inconvenient.

Disclosure of Invention

In view of the above, the present invention is to provide a nucleic acid extracting solution from paraffin section sample and an extracting method thereof, so as to solve the above problems in the prior art.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a nucleic acid extracting solution for a paraffin section sample comprises the following components in parts by mass: 5 to 30 percent of liquid dewaxing agent, 10 to 300mM of Tris-HCl, 1 to 100mM of EDTA, 0.02 to 3 percent of LDS (lithium dodecyl sulfate), 0.05 to 3M of guanidine hydrochloride, 0.05 to 5 percent of N-acetylcysteine, 1 to 30mg/ml of potassium chloride, 0.05 to 1M of sodium chloride, 0.02 to 2M of sodium hydroxide, 0.1 to 10 percent of triton and 0.1 to 5 percent of Tween 20.

Preferably, the components in parts by mass are as follows: 10 to 25 percent of liquid dewaxing agent, 10 to 200mM of Tris-HCl, 1 to 50mM of EDTA, 0.1 to 1 percent of LDS, 0.5 to 1M of guanidine hydrochloride, 0.5 to 2 percent of N-acetylcysteine, 1 to 10mg/ml of potassium chloride, 0.05 to 0.5M of sodium chloride, 0.02 to 1M of sodium hydroxide, 0.1 to 2 percent of triton and 0.1 to 2 percent of Tween 20.

The invention also provides a nucleic acid extraction method for the paraffin section sample, which uses the nucleic acid extracting solution and comprises the following steps: removing redundant paraffin on the periphery of a paraffin section sample, putting the paraffin section sample into a nuclease-free centrifugal tube, adding the nucleic acid extracting solution, putting the nucleic acid extracting solution into a constant-temperature oscillation incubator at 90-98 ℃, oscillating and incubating for 20-60 min, wherein the intermediate clear solution is the extracted nucleic acid, and directly sampling for downstream PCR detection and the like.

Preferably, the method is: removing redundant paraffin on the periphery of the paraffin section sample, putting the paraffin section sample into a centrifugal tube without nuclease, adding the nucleic acid extracting solution, putting the sample into a constant-temperature oscillation incubator at 95 ℃, and carrying out oscillation incubation for 30min, wherein the clear solution of the middle layer is the extracted nucleic acid.

The invention has the following beneficial effects: the nucleic acid extracting solution provided by the invention can provide a strong alkaline environment to promote the release of cell membrane broken nucleic acid, meanwhile, LDS can effectively remove impurities such as protein, N-acetylcysteine can reduce the viscosity of a sample and assist the breakage of cell membranes, multi-step treatment is not needed to remove impurities, the released nucleic acid can be directly used for downstream PCR experiments and the like, organic solvents such as xylene and the like are not needed to be used for dewaxing in the whole extraction process, alcohol reagents are not needed to be used for washing, the harm to human bodies is reduced, meanwhile, complex operations such as dewaxing, cracking liquid, washing, elution and the like are not needed in the whole extraction process, the process loss is reduced, the nucleic acid extracting solution does not need to be matched with a centrifugal machine, an extractor and the like for use, the operation is simple and convenient, the efficiency is high, and the extraction cost is reduced.

Drawings

FIG. 1 is a graph of fluorescent quantitative PCR amplification;

FIG. 2 is an agarose gel electrophoresis of the amplified products of sample Nos. 1 and 4.

Detailed Description

So that the manner in which the features and aspects of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings.

The invention relates to a nucleic acid extracting solution for a paraffin section sample, which comprises the following components in parts by mass: 5 to 30 percent of liquid dewaxing agent, 10 to 300mM of Tris-HCl, 1 to 100mM of EDTA, 0.02 to 3 percent of LDS, 0.05 to 3M of guanidine hydrochloride, 0.05 to 5 percent of N-acetylcysteine, 1 to 30mg/ml of potassium chloride, 0.05 to 1M of sodium chloride, 0.02 to 2M of sodium hydroxide, 0.1 to 10 percent of triton and 0.1 to 5 percent of Tween 20.

The nucleic acid extracting solution provided by the invention can provide a strong alkaline environment to promote the release of cell membrane broken nucleic acid, meanwhile, LDS can effectively remove impurities such as protein, N-acetylcysteine can reduce the viscosity of a sample and assist the cell membrane breakage, impurities are removed without multi-step treatment, and the released nucleic acid can be directly used for downstream PCR and other experiments.

The invention relates to a nucleic acid extraction method for a paraffin section sample, which uses the nucleic acid extracting solution and comprises the following steps: removing redundant paraffin on the periphery of a paraffin section sample, putting the paraffin section sample into a nuclease-free centrifugal tube, adding the nucleic acid extracting solution, putting the nucleic acid extracting solution into a constant-temperature oscillation incubator at 90-98 ℃, oscillating and incubating for 20-60 min, taking out the middle clear solution of the centrifugal tube to obtain the extracted nucleic acid, and directly sampling to perform downstream PCR detection and the like.

Preferably, the method is: removing redundant paraffin on the periphery of a paraffin section sample, putting the paraffin section sample into a nuclease-free centrifuge tube, adding the nucleic acid extracting solution, putting the nucleic acid extracting solution into a constant-temperature oscillation incubator at 95 ℃, oscillating and incubating for 30min, taking out a supernatant liquid in the centrifuge tube to obtain the extracted nucleic acid, and directly sampling to perform downstream PCR detection and the like.

The whole extraction process does not need to use organic solvents such as dimethylbenzene for dewaxing, alcohol reagents are used for washing, harm to a human body is reduced, meanwhile, the whole extraction process does not need to be subjected to complex operations such as dewaxing, cracking liquid, washing and elution, process loss is reduced, the whole extraction process does not need to be matched with a centrifugal machine, an extraction instrument and the like, the operation is simple and convenient, the efficiency is high, and the extraction cost is reduced.

Example 1

A nucleic acid extracting solution for a paraffin section sample comprises the following components in parts by mass: 20% liquid dewaxing agent, 200mM Tris-HCl, 20mM EDTA, 1M guanidine hydrochloride, 1% N-acetylcysteine, 2mg/ml potassium chloride, 0.2M sodium chloride, 0.04M sodium hydroxide, 1% triton and 1% Tween 20.

Example 2

A nucleic acid extracting solution for a paraffin section sample comprises the following components in parts by mass: 20% liquid dewaxing agent, 200mM Tris-HCl, 20mM EDTA, 1% LDS, 1M guanidine hydrochloride, 1% N-acetylcysteine, 2mg/ml potassium chloride, 0.2M sodium chloride, 0.04M sodium hydroxide, 1% triton and 1% Tween 20.

Example 3

A method for extracting nucleic acid from a paraffin section sample utilizes the extracting solution of embodiment 2 to extract, and comprises the following specific steps:

(1) removing redundant paraffin on the periphery of the section sample, and putting the section sample into a nuclease-free 1.5ml centrifuge tube;

(2) adding 500 μ l of nucleic acid extract into a centrifuge tube, and placing into a constant temperature shaking incubator at 95 deg.C for shaking incubation for 30 min;

(3) taking out the centrifuge tube, wherein the intermediate clarified solution is the extracted nucleic acid solution, and directly sampling for downstream detection such as PCR.

Example 4

A method for extracting nucleic acid from paraffin section samples utilizes the extracting solution of embodiment 1 to extract, and comprises the following specific steps:

(2) adding 500 μ l of nucleic acid extract into a centrifuge tube, and placing into a constant temperature shaking incubator (model: VIB 1000) at 95 deg.C for shaking incubation for 30 min;

Comparative example

A kit and a method for extracting paraffin section sample DNA are provided, wherein an Shanghai worker Ezup column type paraffin section genome DNA extraction kit (cargo number B518269) is used for extraction, and the method comprises the following specific steps:

(1) processing a sample, namely adding a paraffin section sample into a 1.5ml centrifuge tube;

(2) add 550. mu.l Buffer FLS to the centrifuge tube, add 50. mu.l Buffer RLA, vortex vigorously for 10 seconds;

(3) water bath at 95-98 deg.C for 30min, and mixing by reversing for 4 times;

(4) centrifuging at 12000 rpm for 5-10 min;

(5) carefully sucking the middle layer water phase and transferring the middle layer water phase into a new centrifuge tube;

(6) adding 2 times of volume of absolute ethyl alcohol into the centrifugal tube, fully and uniformly mixing, and standing for 2-5 minutes at room temperature;

(7) adding the mixed solution obtained in the step (6) into an adsorption column, centrifuging at 8000 rpm for 2min, pouring off waste liquid, and putting the adsorption column back into the collection pipe again;

(8) adding 500 μ l PW Solution into the adsorption column, centrifuging at 8000 rpm for 2min, discarding waste liquid, and placing the adsorption column into the collecting tube;

(9) adding 600 μ l PW Solution into the adsorption column, centrifuging at 8000 rpm for 2min, removing waste liquid, and placing the adsorption column into the collecting tube;

(10) repeating the step (9) once;

(11) the adsorption column was returned to the collection tube and centrifuged at 12000 rpm for 2 min.

(12) Taking out the adsorption column, putting the adsorption column into a new 1.5ml centrifuge tube, adding 30-50 μ l of Elution Buffer into the center of the adsorption membrane, standing for 3 minutes, and centrifuging at 12000 rpm for 2min to obtain the DNA solution.

Taking paraffin section samples, wherein 3, 6, 9 and 12 paraffin section samples are respectively taken as one group, 4 groups are provided in total, the numbers of the 4 groups are 1-4 respectively, the samples in each group are equally divided into 3 parts, and 4 groups of paraffin section samples with equal parts are obtained; after DNA extraction is performed on each group of 3 equal parts of paraffin section samples by the extraction methods in the embodiment 3, the embodiment 4 and the comparative example, DNA extraction solutions are obtained, and after the DNA extraction solutions are diluted into DNA solutions with the same proportion, 5 ul of the amplified DNA solutions is subjected to fluorescent quantitative PCR amplification, the results are shown in the table 1 and the figure 1, and 5 ul of products obtained after the amplification of the samples with the number 1 and the number 4 is subjected to 1% agarose gel electrophoresis detection, the results are shown in the figure 2, and M in the figure 2: DNA Marker; y: negative control; 1-3: examples 3, 4 and comparative example, in that order, sample No. 1; 4-6: examples 3, 4 and comparative example of sample No. 4 were followed.

TABLE 1 fluorescent quantitative PCR amplification results

As can be seen from Table 1, the methods described in examples 3 and 4 can successfully extract nucleic acid from paraffin section samples, and the extracted nucleic acid can meet downstream fluorescent quantitative PCR detection, and the Ct value of nucleic acid extraction amplification in example 3 is about 0.5 earlier than that in example 4, and the main reason is that 1% of LDS is added in the nucleic acid extracting solution in example 3 compared with the extracting solution in example 4, which is beneficial to removing protein impurities and the like, reduces the influence on downstream detection, and enables the nucleic acid extraction purity to be higher; the Ct values of the extracted and amplified nucleic acids of the

embodiments

3 and 4 are advanced compared with the comparative example, the Ct values of the extracted and amplified nucleic acids of the embodiment 3 are advanced by 0.5-1 Ct compared with the comparative example, and the difference of an amplified electrophoretogram and the embodiment 3 is not obvious, but the operation steps of the extraction method are obviously fewer than the comparative example, the extraction process does not need to pass through complicated extraction steps, and the whole extraction process does not need to use machines such as a centrifuge and the like; in addition, the extraction time of the invention is 30 minutes, which saves 20-30 minutes compared with the comparison (about 50-60 minutes), and can greatly reduce the time cost; compared with the comparative example, the invention does not need to use various reagent components such as washing, elution and the like, and can effectively save the reagent cost.

The specific type of the above-mentioned devices is not limited and detailed, and the deep connection mode of the above-mentioned devices is not detailed, and can be understood by those skilled in the art as the common general knowledge.

The above description is only exemplary of the present invention and should not be taken as limiting the scope of the present invention, and any modifications, equivalents, improvements, etc. that are within the spirit and principle of the present invention should be included in the present invention.

Claims

1. The nucleic acid extracting solution for the paraffin section sample is characterized by comprising the following components in parts by mass: 5 to 30 percent of liquid dewaxing agent, 10 to 300mM of Tris-HCl, 1 to 100mM of EDTA, 0.02 to 3 percent of LDS, 0.05 to 3M of guanidine hydrochloride, 0.05 to 5 percent of N-acetylcysteine, 1 to 30mg/ml of potassium chloride, 0.05 to 1M of sodium chloride, 0.02 to 2M of sodium hydroxide, 0.1 to 10 percent of triton and 0.1 to 5 percent of Tween 20.

2. The nucleic acid extracting solution for paraffin section samples according to claim 1, wherein the components in parts by mass are: 10 to 25 percent of liquid dewaxing agent, 10 to 200mM of Tris-HCl, 1 to 50mM of EDTA, 0.1 to 1 percent of LDS, 0.5 to 1M of guanidine hydrochloride, 0.5 to 2 percent of N-acetylcysteine, 1 to 10mg/ml of potassium chloride, 0.05 to 0.5M of sodium chloride, 0.02 to 1M of sodium hydroxide, 0.1 to 2 percent of triton and 0.1 to 2 percent of Tween 20.

3. A method for extracting nucleic acid from a paraffin section sample, which comprises using the nucleic acid extract according to claim 1 or 2, comprising: removing redundant paraffin on the periphery of the paraffin section sample, putting the paraffin section sample into a nuclease-free centrifugal tube, adding the nucleic acid extracting solution, putting the nucleic acid extracting solution into a constant-temperature oscillation incubator at 90-98 ℃, and performing oscillation incubation for 20-60 min, wherein the intermediate clarified solution is the extracted nucleic acid.

4. The method for extracting nucleic acid from a paraffin section sample according to claim 3, wherein the method comprises: removing redundant paraffin on the periphery of the paraffin section sample, putting the paraffin section sample into a centrifugal tube without nuclease, adding the nucleic acid extracting solution, putting the sample into a constant-temperature oscillation incubator at 95 ℃, and carrying out oscillation incubation for 30min, wherein the clear solution of the middle layer is the extracted nucleic acid.