CN114891778A

CN114891778A - Method and kit for full-automatic extraction of DNA of paraffin-embedded tissue section

Info

Publication number: CN114891778A
Application number: CN202210352752.9A
Authority: CN
Inventors: 朱小娟; 陈文�; 徐骏; 吴志铭
Original assignee: Hangzhou Danwei Biotechnology Co ltd
Current assignee: Hangzhou Danwei Biotechnology Co ltd
Priority date: 2022-03-31
Filing date: 2022-03-31
Publication date: 2022-08-12

Abstract

The invention relates to a method and a kit for full-automatic extraction of DNA of a paraffin-embedded tissue section, wherein the method comprises the following steps: s1, preparing a paraffin-embedded tissue section; s2, adding cell lysate, proteinase K solution and liquid paraffin to the paraffin-embedded tissue section; s3, adding super-cis nano magnetic beads, a cleaning solution and an eluent into the system of S2 according to a nucleic acid extraction program; s4, extracting nucleic acid; and S5, finishing extraction. In general, liquid paraffin does not serve as a component of a reagent for nucleic acid extraction from paraffin-embedded tissues. The invention uses the liquid paraffin as the extracting agent, and can extract the melted solid paraffin into the liquid paraffin and mix the solid paraffin, thereby reducing the melting point of the paraffin, keeping the paraffin in a liquid state when the paraffin is cooled, and being beneficial to the subsequent automatic operation of nucleic acid extraction.

Description

Method and kit for full-automatic extraction of DNA of paraffin-embedded tissue section

Technical Field

The invention relates to the field of molecular biology, in particular to a method and a kit for fully automatically extracting DNA of a paraffin-embedded tissue section.

Background

Formalin-fixed paraffin-embedded tissues are often used for the study and diagnosis of tumor cases as one of the most convenient sources of clinical material. The molecular level gene detection and screening using paraffin tissue sections as materials has become a routine means for clinical diagnosis and related research. The extraction of nucleic acid from paraffin-embedded tissues is the daily work of clinical cases, and the currently commonly used paraffin sample nucleic acid extraction kit adopts xylene or other dewaxing solutions for dewaxing, protease digestion of protein to release nucleic acid, and then purification by a nucleic acid purification column chromatography or a nano magnetic bead method to obtain nucleic acid.

However, in the current methods used in laboratory, no matter the nucleic acid is purified by column-passing method or nano-magnetic bead method, the dewaxing operation step and the cleaning of the dewaxing solution are difficult to discard, and thus the full-flow operation cannot be effectively performed in the automated equipment. The reason for this is mainly that the paraffin for embedding is liable to solidify in a cooled state and the dewaxing solution needs to be repeatedly washed. Because the real high-efficient automation can not be reached, cause the paraffin embedding tissue nucleic acid extraction process to waste time, hard and cause the problem of operation repeatability deviation easily.

Disclosure of Invention

The invention aims to solve the technical problems of overcoming the defects in the prior art, providing a full-automatic extraction method of paraffin-embedded tissue section DNA, and solving the defects of difficult automation, long operation time, high personnel requirement and the like in the prior art of paraffin-embedded tissue section DNA extraction.

Based on the purpose, the invention adopts the following technical scheme:

a method for fully automatically extracting DNA of a paraffin-embedded tissue section comprises the following steps:

s1, preparation of paraffin-embedded tissue sections: placing the paraffin embedded tissue on a paraffin slicer for slicing to obtain a tissue slice with the slice thickness of 3-15 mu M; floating the sliced tissues on 45 +/-5 ℃ warm water in a spreading machine, flattening the tissues, taking out the tissues to be extracted by using a glass slide, putting the tissues into an oven for baking, and taking out the tissues after moisture is dried to obtain paraffin embedded tissue slices to be detected;

s2, adding cell lysate, proteinase K solution and liquid paraffin to the paraffin-embedded tissue section;

s3, adding superparamagnetic beads, a cleaning solution and an eluent into the system of S2 according to a nucleic acid extraction program;

s4, placing the sample in an automatic nucleic acid extractor (magnetic bead method), setting a nucleic acid extraction program, and starting automatic operation of nucleic acid extraction;

and S5, finishing the extraction, and carrying out the next operation by using the eluent containing the extracted product or transferring the eluent to an enzyme-free storage tube for storage.

In general, liquid paraffin does not serve as a component of a reagent for nucleic acid extraction from paraffin-embedded tissues. The invention uses the liquid paraffin as the extracting agent, and can extract the melted solid paraffin into the liquid paraffin and mix the solid paraffin, thereby reducing the melting point of the paraffin, keeping the paraffin in a liquid state when the paraffin is cooled, and being beneficial to the subsequent automatic operation of nucleic acid extraction.

Preferably, the cell lysate contains guanidine hydrochloride or guanidine isothiocyanate at a concentration of 2-5M in step S2, and the pH of the cell lysate is 6.0-7.5.

Preferably, the volume of the liquid paraffin used in the step S2 is 600 to 900. mu.L/600. mu.L of cell lysate.

Preferably, the nucleic acid extraction procedure of step S4 is:

preferably, the S3 superparamagnetic nano-beads are silicon hydroxyl superparamagnetic nano-beads, and the particle size is 100-300 nm.

Preferably, the washing solution of S3 comprises washing solution 1 and washing solution 2, wherein washing solution 1 comprises 16. mu. L0.5M Tris, 4.7mgNaCl, 300. mu.L absolute ethyl alcohol and 180. mu.L purified water, and washing solution 2 comprises 500. mu.L 75 vol% ethanol.

Preferably, the elution reagent in S3 is TE buffer.

A kit for full-automatic extraction of DNA of paraffin-embedded tissue sections comprises: 600. mu.L of a cell lysate containing 2 to 5M guanidine hydrochloride or guanidine isothiocyanate and having a pH of 6.0 to 7.5, 20. mu.L of 20mg/mL proteinase K solution,

600 to 900 μ L of liquid paraffin;

washing solution 1 consisting of 16. mu. L0.5M Tris, 4.7mgNaCl, 300. mu.L absolute ethanol, 180. mu.L purified water, washing solution 2 consisting of 500. mu.L 75% (by volume) ethanol.

Preferably, the cell lysate is prepared by mixing 300. mu.L of 4-5M guanidine hydrochloride or guanidine isothiocyanate with 300. mu.L of isopropanol.

Compared with the existing paraffin-embedded tissue nucleic acid extraction technology, the invention has the beneficial effects that:

1. the cleaning operation of dewaxing and dewaxing liquid is not needed;

2. the nucleic acid extraction and purification process is easy to operate automatically, the requirements on operators are reduced, and the extraction efficiency and the extraction repeatability are improved;

drawings

FIG. 1 is a schematic view of the process for fully automatically extracting DNA from paraffin-embedded tissue sections according to the present invention;

FIG. 2 is a graph showing the fluorescence PCR detection and amplification curve of DNA (of the present invention) extracted from paraffin-embedded lung cancer tissue sections;

FIG. 3 is a graph showing the fluorescence PCR detection and amplification curve of DNA extracted from a lung cancer paraffin-embedded tissue section by a conventional method (purchased commercial reagent);

FIG. 4 is a fluorescent PCR detection and amplification curve diagram of DNA (of the invention) fully automatically extracted from paraffin-embedded colorectal cancer tissue section;

FIG. 5 is a graph showing the fluorescence PCR detection and amplification curve of DNA extracted from paraffin-embedded colon cancer tissue section by conventional method (reagent for outsourcing commercial products).

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.

In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.

Outsourcing of commercial reagents:

the silicon hydroxyl modified superparamagnetic nano-bead has the particle size of 150nm and is purchased from Suzhou degree biotechnology limited;

paraffin embedded tissue RNA rapid extraction kit (centrifugal column type), beijing Baitaike biotechnology limited.

Example 1

A method for fully automatically extracting DNA of a paraffin-embedded tissue section is shown in figure 1, and comprises the following specific steps:

1. preparation of nucleic acid extraction and purification reagent

(1) Preparation of cell lysate

Each cell lysate is prepared by evenly mixing 300 mu L of 4-5M guanidinium isothiocyanate buffer solution and 300 mu L of isopropanol, and the pH value is 7.3 +/-0.2.

(2) Preparation of cleaning solution 1

Each wash1 consisted of 16. mu. L0.5M Tris, 4.7mgNaCl, 300. mu.L absolute ethanol, 180. mu.L purified water.

(3) Preparation of cleaning solution 2

Each wash2 consisted of 500. mu.L of 75% ethanol.

(4) Preparation of the eluent

Each aliquot consisted of 100. mu.L of TE buffer.

(5) Subpackaging each component reagent to 96 pore plates

2. Nucleic acid extraction

5 cases of lung cancer paraffin-embedded tissues and 5 cases of colorectal cancer paraffin-embedded tissues were subjected to three groups of treatments as follows:

(1) placing the paraffin embedded tissue on a paraffin slicer for slicing to obtain a tissue slice with the thickness of 5 mu M; floating the sliced tissues on warm water in a spreading machine, flattening the tissues, taking out the tissues to be extracted by using a glass slide, putting the tissues into an oven for baking, and taking out the tissues after moisture is dried.

4 sections were prepared for each sample.

(2) Treatment group-automatic extraction group (method of the invention)

Two paraffin sections were transferred into the lysate well and 20 μ L (20mg/mL) of proteinase K solution was added;

add 600. mu.L of liquid paraffin to the lysate well.

A pre-sealing plate is placed in an adaptive automatic nucleic acid extraction and purification instrument (Hangzhou Osheng Pure-32A), a magnetic needle sleeve starting device is installed, and after an experiment configuration interface is entered, an extraction program is set (see table 1).

TABLE 1 this extraction program settings

Step (ii) of

Hole site

Step (ii) of

Standing time

Time of mixing

Time of magnetic attraction

Volume of

Temperature state

Temperature of

1

Cell lysis

45 minutes

0

1000μL

Heating of

65℃

2

1

De-crosslinking incubation

90 minutes

0

1000μL

Heating of

92℃

3

2

Get magnetism and add magnetism

/

1 minute

60 seconds

500μL

Without heating

/

4

1

Nucleic acid binding

/

10 minutes

60 seconds

1000μL

Without heating

65℃

5

3

First cleaning

/

60 seconds

500μL

Without heating

/

6

4

Second cleaning

/

60 seconds

500μL

Without heating

/

7

5

Third cleaning

/

60 seconds

500μL

Without heating

/

8

6

Magnetic bead air drying

2 minutes

/

Without heating

/

9

6

Nucleic acid elution

/

120 seconds

60 seconds

100μL

Heating of

65℃

10

1

Abandon magnetic bead

/

5 seconds

0

1000μL

Without heating

/

The program is started to start the nucleic acid extraction operation. Transferring the extracted nucleic acid into an enzyme-free centrifugal tube;

the nucleic acid concentration and purity of each extracted product was determined using an ultraviolet spectrophotometer.

(3) Control group-semi-automatic extraction group (commercial reagent)

Adding 1ml xylene into a centrifugal tube filled with FFPE tissue, uniformly mixing by vortex oscillation, and incubating at 50 ℃ for 3min to melt paraffin;

the sample was centrifuged at 10,000g for 2min at room temperature, with xylene carefully discarded;

1ml of absolute ethanol was added and vortexed for 10 seconds. Centrifuge at 10,000g for 2min at room temperature. Carefully discard the ethanol. Taking care not to aspirate FFPE tissue fragments;

adding 1ml of absolute ethyl alcohol again, and carrying out vortex oscillation for 10 seconds; centrifuge at 10,000g for 2min at room temperature. Carefully removing the ethanol by suction; centrifuging at high speed for a short time, and sucking and removing residual ethanol as much as possible;

and opening the centrifugal tube cover, standing at room temperature or 37 ℃ for 10-15 min, and airing residual ethanol.

Adding 200 mu L of digestive juice and 20 mu L of proteinase K, carrying out vortex oscillation for 10 seconds, and re-suspending the dewaxed FFPE tissue slice;

placing the tissue slices in a 56 ℃ water bath for incubation for 1-3 hours, oscillating at intervals, and performing the following operations when the tissue slices are completely cracked and disappear; does not affect DNA extraction.

Standing and incubating for 1 hour in a water bath at 90 ℃;

the liquid in the tube was collected by brief centrifugation and equilibrated to room temperature. The solution was transferred to pre-closed plates (8T/cassette) at

column

2 and 8 lysate locations for extraction.

Placing a pre-sealing plate in an adaptive automatic nucleic acid extraction and purification instrument (LunAmple X48), installing a magnetic needle sleeve starting device, and setting an extraction program after entering an experimental configuration interface:

step 1: naming step (transfer of magnetic beads); working position 1, working volume 900ul, movement mode 2 (times 1, time 10 s);

step 2: naming step (lysis LB); working position 2, working volume 700ul, movement mode 3 (magnetic bead mixing: frequency 2, time 600s, speed medium speed; magnetic bead adsorption: frequency 1, time 10s), temperature is set to 65 ℃ in this step;

and step 3: a naming step (Wash 1); working position 1, working volume 900ul, movement mode 3 (magnetic bead mixing: frequency 1, time 180s, speed rapid; magnetic bead adsorption: frequency 1, time 5 s);

and 4, step 4: a naming step (Wash 2); working position 3, working volume 900ul, movement mode 3 (magnetic bead mixing: frequency 1, time 180s, speed rapid; magnetic bead adsorption: frequency 1, time 5 s);

and 5: a naming step (Wash 3); a working position 4, a working volume of 500ul, and a motion mode 3 (magnetic bead mixing: frequency of 1, time of 180s, speed of rapid mixing; magnetic bead adsorption: frequency of 1, time of 5 s);

step 6: a naming step (Wash 4); working position 5, working volume 500ul, movement mode 3 (magnetic bead mixing: frequency 1, time 180s, speed rapid; magnetic bead adsorption: frequency 1, time 5 s);

and 7: naming step (elution of EB); the working position 6, the working volume 50ul-100ul, the movement mode 3 (action delay 180s, magnetic bead mixing times 1, time 600s, speed rapid, magnetic bead adsorption times 2, time 10s), the temperature of the step is set to 65 ℃;

and 8: a naming step (end); working position 3, working volume 600uL, movement mode 4.

3. Preparation of human EGFR gene detection reagent (fluorescent PCR method)

(1) PCR reaction solutions were prepared as per table 2:

TABLE 2 PCR reaction solution

(2) Subpackaging PCR reaction solution

The PCR reaction solution was dispensed into a PCR tube at a volume of 20. mu.L/reaction.

(3) Nucleic acid addition

Adding the extracted nucleic acid into a PCR tube according to 5 mu L/sample, covering the PCR tube with a cover, shaking and mixing uniformly, and performing instantaneous centrifugation on the PCR tube and standing on the PCR tube.

4. Detection on machine

Placing the PCR tube into a fluorescent PCR instrument for fluorescent PCR amplification, wherein the detection program comprises the following steps: maintaining at 50 deg.C for 2 min; maintaining at 95 deg.C for 1 min; (95 ℃ for 10 s; 60 ℃ for 31s and fluorescence collected) 40 cycles. After the operation of the equipment detection program is finished, observing an amplification curve graph, and reading a Ct value;

5. the result of the detection

The results of detection (fluorescence curve Ct values) for each treatment group are shown in tables 3 and 4 and FIGS. 2 to 5. FIG. 2 is a graph showing the fluorescence PCR detection and amplification curve of DNA (of the present invention) extracted from paraffin-embedded lung cancer tissue sections; FIG. 3 is a fluorescence PCR detection and amplification curve diagram of DNA extracted by a conventional method (paraffin-embedded tissue RNA rapid extraction kit (centrifugal column type), Beijing Baitag) for lung cancer paraffin-embedded tissue section DNA; FIG. 4 is a graph showing the fluorescence PCR detection and amplification curve of DNA (of the present invention) extracted from paraffin-embedded colorectal cancer tissue sections in a fully automatic manner; FIG. 5 is a fluorescence PCR detection amplification curve diagram of DNA extracted by a conventional method (paraffin-embedded tissue RNA rapid extraction kit (centrifugal column type), Beijing Baitaike) for colon cancer paraffin-embedded tissue section DNA.

TABLE 3 determination of concentration and purity of extracted product

TABLE 4 Ct values of fluorescence PCR detection results

Table 3 shows that the method for fully automatically extracting DNA from paraffin-embedded tissue sections (the present invention) has no significant difference in purity of the extracted nucleic acids compared to the conventional extraction method (extraction after dewaxing), but the method of the present invention is slightly superior to the conventional extraction method (extraction after dewaxing) in concentration. The same conclusions were also drawn from the results of the fluorescence PCR measurements shown in Table 4 and FIGS. 2-5.

EXAMPLE 2 kit

A reagent box for full-automatic extraction of paraffin-embedded tissue section DNA comprises the following components:

600. mu.L of cell lysate with pH 7.0,

20 μ L of 20mg/mL proteinase K solution,

600 μ L of liquid paraffin;

The cell lysate is prepared by uniformly mixing 300 mu L of 5M guanidinium isothiocyanate and 300 mu L of isopropanol.

The embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same or similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.

The method and the kit for fully automatically extracting the DNA of the paraffin-embedded tissue section are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, it is possible to make various improvements and modifications to the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

1. A method for fully automatically extracting DNA of a paraffin-embedded tissue section is characterized by comprising the following steps:

s4, placing the sample in an automatic nucleic acid extractor, setting a nucleic acid extraction program, and starting automatic operation of nucleic acid extraction;

2. The method for full-automatic extraction of DNA from paraffin-embedded tissue sections according to claim 1, characterized in that: s2 step, the cell lysis solution contains 2-5M guanidine hydrochloride or guanidine isothiocyanate, and the pH value of the cell lysis solution is 6.0-7.5.

3. The method for full-automatic extraction of DNA from paraffin-embedded tissue sections according to claim 1, characterized in that: the volume dosage of the liquid paraffin in the step S2 is 600 mu L to 900 mu L per 600 mu L of cell lysate.

4. The method for full-automatic extraction of DNA from paraffin-embedded tissue sections according to claim 1, characterized in that: the nucleic acid extraction procedure of step S4 is:

5. the method for full-automatic extraction of DNA from paraffin-embedded tissue sections according to claim 1, characterized in that: the super-cis nano magnetic bead of S3 is a silicon hydroxyl super-cis nano magnetic bead with the particle size of 100-300 nm.

6. The method for full-automatic extraction of DNA from paraffin-embedded tissue sections according to claim 1, characterized in that: the cleaning solution of S3 is composed of cleaning solution 1 and cleaning solution 2, wherein,

washing solution 1 consisted of 16. mu. L0.5M Tris, 4.7mg NaCl, 300. mu.L absolute ethanol, 180. mu.L purified water, and washing solution 2 consisted of 500. mu.L 75% (by volume) ethanol.

7. The method for full-automatic extraction of DNA from paraffin-embedded tissue sections according to claim 1, characterized in that: the eluent in S3 is TE buffer.

8. The full-automatic extraction kit for the DNA of the paraffin-embedded tissue section is characterized by comprising: 600. mu.L of a cell lysate containing 2 to 5M guanidine hydrochloride or guanidine isothiocyanate and having a pH of 6.0 to 7.5, 20. mu.L of a 20mg/mL proteinase K solution,

600 to 900 μ L of liquid paraffin;

cleaning solution 1 consisting of 16 mu L0.5M Tris, 4.7mgNaCl, 300 mu L absolute ethyl alcohol and 180 mu L purified water,

wash2 consisting of 500. mu.L of 75% by volume ethanol.

9. The kit for full-automatic extraction of DNA from paraffin-embedded tissue sections according to claim 8, characterized in that: the cell lysate is prepared by uniformly mixing 300 mu L of 4-5M guanidine hydrochloride or guanidine isothiocyanate and 300 mu L of isopropanol.