CN117737051A - Full-automatic extraction method of paraffin-embedded tissue slice DNA - Google Patents
Full-automatic extraction method of paraffin-embedded tissue slice DNA Download PDFInfo
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- CN117737051A CN117737051A CN202311809927.5A CN202311809927A CN117737051A CN 117737051 A CN117737051 A CN 117737051A CN 202311809927 A CN202311809927 A CN 202311809927A CN 117737051 A CN117737051 A CN 117737051A
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- paraffin
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- 238000000605 extraction Methods 0.000 title claims abstract description 46
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 28
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 28
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 28
- 239000012188 paraffin wax Substances 0.000 claims abstract description 25
- 239000003480 eluent Substances 0.000 claims abstract description 14
- 239000011324 bead Substances 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 238000004140 cleaning Methods 0.000 claims abstract description 10
- 229940057995 liquid paraffin Drugs 0.000 claims abstract description 10
- 108091005804 Peptidases Proteins 0.000 claims abstract description 8
- 239000004365 Protease Substances 0.000 claims abstract description 8
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 8
- 239000011521 glass Substances 0.000 claims abstract description 8
- 238000003892 spreading Methods 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 23
- 239000006166 lysate Substances 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims description 4
- 238000003860 storage Methods 0.000 claims description 4
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 3
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000008213 purified water Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 7
- 238000000746 purification Methods 0.000 abstract description 4
- 238000007789 sealing Methods 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 238000005336 cracking Methods 0.000 abstract 2
- 238000004321 preservation Methods 0.000 abstract 2
- 235000019419 proteases Nutrition 0.000 description 5
- 238000007400 DNA extraction Methods 0.000 description 3
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- 230000003044 adaptive effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
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Abstract
The invention discloses a full-automatic extraction method of paraffin-embedded tissue slice DNA, which comprises the following steps of S1, embedding paraffin into a tissue slice, putting the tissue slice into a slice spreading machine, taking out the tissue by a glass slide, putting the tissue into a baking oven for baking after moisture, taking out the tissue to prepare a slice, S2, transferring the paraffin slice into a cracking liquid hole, adding protease solution and liquid paraffin into the cracking liquid, and S3: adding nano magnetic beads, cleaning liquid and eluent into S2, putting a pre-sealing plate into an automatic nucleic acid extraction and purification instrument, installing a magnetic needle sleeve starting device, setting an extraction program, starting the nucleic acid extraction operation, and after S5 extraction is finished, carrying out the next operation on the eluent containing the extracted product or transferring the eluent into an enzyme-free preservation tube for preservation, and measuring the nucleic acid concentration and purity of the extract by utilizing a photometer, so that a dewaxing reagent is not required, thereby avoiding contact with toxic components in the dewaxing reagent, and automatically finishing the extraction.
Description
Technical Field
The invention relates to the technical field of DNA extraction, in particular to a full-automatic extraction method of paraffin embedded tissue slice DNA.
Background
DNA extraction of samples is mainly a CTAB method, and other methods include physical methods such as a glass bead method, an ultrasonic method, a grinding method and a freeze thawing method. Chemical means such as guanidine isothiocyanate and alkaline cleavage. Biological mode: enzymatic methods. Depending on the manner of nucleic acid isolation and purification, there are siliceous materials, anion exchange resins, and the like.
At present, a plurality of times of manual participation are needed in the paraffin embedded tissue sample treatment process, so that the laboratory efficiency is affected, and the repeatability and uniformity of manual operation are unstable; and the dewaxing reagent used in the traditional paraffin embedded tissue sample nucleic acid extraction process has toxicity.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce a few preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.
The present invention has been made in view of the above and/or problems associated with the prior art methods for the fully automated extraction of DNA from paraffin-embedded tissue sections.
Therefore, the invention aims to provide a full-automatic extraction method of paraffin embedded tissue slice DNA, when in use, paraffin embedded tissue is put on a paraffin microtome for slicing, and tissue slices with the slice thickness of 3-15 mu M are obtained; the sliced tissues are floated on warm water in a slice spreading machine, the tissues are flattened, the tissues to be extracted are fished up by using a glass slide, the tissues are put into an oven for baking, the tissues are taken out after moisture is dried, and 4 slices are prepared for each sample. Two paraffin sections were transferred into lysate wells and 20. Mu.L (20 mg/mL) of proteinase K solution was added; liquid paraffin 600. Mu.L was added to the lysate well. And placing a pre-sealing plate in the adaptive automatic nucleic acid extraction and purification instrument, installing a magnetic needle sleeve starting device, and setting an extraction program after entering an experimental configuration interface. The procedure is started and the nucleic acid extraction operation is started. After the extraction is completed, the nucleic acid is transferred into an enzyme-free centrifuge tube.
In order to solve the technical problems, according to one aspect of the present invention, the following technical solutions are provided:
a method for full-automatic extraction of paraffin-embedded tissue slice DNA, comprising:
s1: firstly, placing paraffin embedded tissues on a paraffin microtome for slicing, and obtaining tissue slices with the slice thickness of 3-15 mu M; floating the sliced tissue on warm water at 45+/-5 ℃ in a piece spreading machine, flattening the tissue, fishing out the tissue to be extracted by using a glass slide, putting the tissue into a baking oven for baking, taking out the tissue after the moisture is dried, and preparing 4 slices for each sample;
s2: transferring the two paraffin sections into a lysate hole, and adding protease solution and liquid paraffin into the lysate hole;
s3: adding nano magnetic beads, cleaning liquid and eluent into the S2;
s4: placing the sample in an automatic nucleic acid extraction instrument, setting a nucleic acid extraction program, and starting automatic operation of nucleic acid extraction;
s5: after the extraction is completed, the eluent containing the extracted product is subjected to the next operation or is transferred to no enzyme
The extract was stored in a storage tube and the nucleic acid concentration and purity of the extract were measured by a photometer.
As a preferred scheme of the method for fully automatically extracting the paraffin embedded tissue slice DNA, the invention is characterized in that the lysate is prepared from 300 mu L of 4-5M guanidine hydrochloride or guanidine isothiocyanate and 300 mu L of isopropanol.
As a preferred embodiment of the method for full-automatic extraction of DNA from paraffin-embedded tissue sections according to the present invention, the protease solution in the step S2 is used in an amount of 20. Mu.L and 20mg/mL, and the liquid paraffin is used in an amount of 600. Mu.L to 900. Mu.L.
As a preferable scheme of the method for fully automatically extracting the DNA of the paraffin embedded tissue slice, the nano magnetic beads are silicon hydroxyl super-cis nano magnetic beads with the particle size of 100nm to 300 nm.
As a preferred scheme of the method for fully automatically extracting the paraffin-embedded tissue slice DNA, in the step S3, the cleaning solution consists of a cleaning solution 1 and a cleaning solution 2, wherein the cleaning solution 1 consists of 16 mu L of 0.5M Tris, 4.7mg NaCl, 300 mu L of absolute ethyl alcohol and 180 mu L of purified water, and the cleaning solution 2 consists of 500 mu L of 75% ethyl alcohol.
As a preferable scheme of the method for full-automatic extraction of paraffin-embedded tissue slice DNA, in the step S3, the eluent is TE buffer solution.
As a preferred scheme of the method for full-automatic extraction of paraffin-embedded tissue slice DNA according to the present invention, the extraction procedure in step S4 is as follows:
。
compared with the prior art, the invention has the following beneficial effects:
1. no dewaxing reagent is required, thereby avoiding contact with toxic components in the dewaxing reagent. 2. The manual operation steps are fewer, the DNA extraction process is fully automated, and the extraction efficiency is effectively improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following detailed description will be given with reference to the accompanying drawings and detailed embodiments, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained from these drawings without inventive faculty for a person skilled in the art. Wherein:
FIG. 1 is a flow chart of a method for full-automatic extraction of paraffin-embedded tissue slice DNA according to the present invention;
FIG. 2 is a flow chart of a method for full-automatic extraction of DNA from paraffin-embedded tissue slices according to the invention;
FIG. 3 is a schematic diagram of the structure of a reagent strip used in the method for full-automatic extraction of DNA from paraffin-embedded tissue sections according to the present invention;
FIG. 4 is a schematic diagram showing the structure of a reagent strip clamped into a full-automatic nucleic acid extractor for a method for full-automatic extraction of DNA of paraffin-embedded tissue sections.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
In the following detailed description of the embodiments of the present invention, the cross-sectional view of the device structure is not partially enlarged to a general scale for the convenience of description, and the schematic is merely an example, which should not limit the scope of the present invention. In addition, the three-dimensional dimensions of length, width and depth should be included in actual fabrication.
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.
Fig. 1 to 4 are schematic views showing the overall structure of an embodiment of a method for full-automatic extraction of DNA from paraffin-embedded tissue sections according to the present invention, referring to fig. 1 to 4, the main body of the method for full-automatic extraction of DNA from paraffin-embedded tissue sections according to the present embodiment includes: s1, S2, S3, S4 and S5.
Example 1
S1: firstly, placing paraffin embedded tissues on a paraffin microtome for slicing, and obtaining tissue slices with the slice thickness of 3-15 mu M; floating the sliced tissue on warm water at 45+/-5 ℃ in a piece spreading machine, flattening the tissue, fishing out the tissue to be extracted by using a glass slide, putting the tissue into a baking oven for baking, taking out the tissue after the moisture is dried, and preparing 4 slices for each sample;
s2: transferring the two paraffin sections into a lysate hole, and adding protease solution and liquid paraffin into the lysate hole;
s3: adding nano magnetic beads, cleaning liquid and eluent into the S2;
s4: placing the sample in an automatic nucleic acid extraction instrument, setting a nucleic acid extraction program, and starting automatic operation of nucleic acid extraction;
s5: after the extraction is completed, the eluent containing the extracted product is subjected to the next operation or is transferred to no enzyme
The extract was stored in a storage tube and the nucleic acid concentration and purity of the extract were measured by a photometer.
Example 2
S1: firstly, placing paraffin embedded tissues on a paraffin microtome for slicing, and obtaining tissue slices with the slice thickness of 3-15 mu M; floating the sliced tissue on warm water at 45+/-5 ℃ in a piece spreading machine, flattening the tissue, fishing out the tissue to be extracted by using a glass slide, putting the tissue into a baking oven for baking, taking out the tissue after the moisture is dried, and preparing 4 slices for each sample;
s2: transferring the two paraffin sections into a lysate hole, and adding protease solution and liquid paraffin into the lysate hole; the lysate is prepared from 300. Mu.L of 4-5M guanidine hydrochloride or guanidine isothiocyanate and 300. Mu.L of isopropanol; the amount of the protease solution in the step S2 is 20 mu L of 20mg/mL, and the amount of the liquid paraffin is 600 mu L to 900 mu L;
s3: adding nano magnetic beads, cleaning liquid and eluent into the S2; the nanometer magnetic beads are silicon hydroxyl super-cis nanometer magnetic beads with the particle size of 100nm to 300 nm; the washing liquid consists of washing liquid 1 and washing liquid 2, and washing liquid 1 consists of 16. Mu.L of 0.5M Tris, 4.7mg NaCl, 300. Mu.L of absolute ethyl alcohol and 180. Mu.L of purified water, and washing liquid 2 consists of 500. Mu.L of 75% ethyl alcohol; the eluent is TE buffer;
s4: placing the sample in an automatic nucleic acid extraction instrument, setting a nucleic acid extraction program, and starting automatic operation of nucleic acid extraction;
wherein, the extraction procedure is:
；
s5: after the extraction is completed, the eluent containing the extracted product is subjected to the next operation or is transferred to no enzyme
The extract was stored in a storage tube and the nucleic acid concentration and purity of the extract were measured by a photometer.
Referring to fig. 1 and 4, in the method for full-automatic extraction of paraffin-embedded tissue slice DNA according to the present embodiment, when the method is specifically used, paraffin-embedded tissue is first placed on a paraffin microtome for slicing, and a tissue slice with a slice thickness of 3-15 μm is obtained; the sliced tissues are floated on warm water in a slice spreading machine, the tissues are flattened, the tissues to be extracted are fished up by using a glass slide, the tissues are put into an oven for baking, the tissues are taken out after moisture is dried, and 4 slices are prepared for each sample. Two paraffin sections were transferred into lysate wells and 20. Mu.L (20 mg/mL) of proteinase K solution was added; liquid paraffin 600. Mu.L was added to the lysate well. And placing a pre-sealing plate in the adaptive automatic nucleic acid extraction and purification instrument, installing a magnetic needle sleeve starting device, and setting an extraction program after entering an experimental configuration interface. Starting the procedure, starting the nucleic acid extraction operation, and determining the nucleic acid concentration and purity of the extract using a photometer, and transferring the extract into an enzyme-free centrifuge tube only when the concentration and purity are acceptable.
Although the invention has been described hereinabove with reference to embodiments, various modifications thereof may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In particular, the features of the disclosed embodiments may be combined with each other in any manner as long as there is no structural conflict, and the exhaustive description of these combinations is not given in this specification merely for the sake of omitting the descriptions and saving resources. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (7)
1. A method for full-automatic extraction of paraffin-embedded tissue slice DNA, comprising:
s1: firstly, placing paraffin embedded tissues on a paraffin microtome for slicing, and obtaining tissue slices with the slice thickness of 3-15 mu M; floating the sliced tissue on warm water at 45+/-5 ℃ in a piece spreading machine, flattening the tissue, fishing out the tissue to be extracted by using a glass slide, putting the tissue into a baking oven for baking, taking out the tissue after the moisture is dried, and preparing 4 slices for each sample;
s2: transferring the two paraffin sections into a lysate hole, and adding protease solution and liquid paraffin into the lysate hole;
s3: adding nano magnetic beads, cleaning liquid and eluent into the S2;
s4: placing the sample in an automatic nucleic acid extraction instrument, setting a nucleic acid extraction program, and starting automatic operation of nucleic acid extraction;
s5: after the extraction is completed, the eluent containing the extracted product is subjected to the next operation or is transferred to no enzyme
The extract was stored in a storage tube and the nucleic acid concentration and purity of the extract were measured by a photometer.
2. The method for full-automatic extraction of paraffin-embedded tissue slice DNA according to claim 1, wherein the lysate is prepared from 300. Mu.L of 4-5M guanidine hydrochloride or guanidine isothiocyanate and 300. Mu.L of isopropanol.
3. The method according to claim 1, wherein the protease solution is used in an amount of 20. Mu.L/mL and the liquid paraffin is used in an amount of 600. Mu.L to 900. Mu.L in step S2.
4. The method for full-automatic extraction of paraffin-embedded tissue slice DNA according to claim 1, wherein in step S3, the nano magnetic beads are silica-hydroxyl super-clockwise nano magnetic beads with a particle size of 100nm to 300 nm.
5. The method according to claim 1, wherein in the step S3, the washing solution is composed of washing solution 1 and washing solution 2, and washing solution 1 is composed of 16 μl of 0.5M Tris, 4.7mg NaCl, 300 μl of absolute ethanol, 180 μl of purified water, and washing solution 2 is composed of 500 μl of 75% ethanol.
6. The method according to claim 1, wherein in step S3, the eluent is TE buffer.
7. The method according to claim 4, wherein the extraction procedure in step S4 is as follows:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311809927.5A CN117737051A (en) | 2023-12-27 | 2023-12-27 | Full-automatic extraction method of paraffin-embedded tissue slice DNA |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311809927.5A CN117737051A (en) | 2023-12-27 | 2023-12-27 | Full-automatic extraction method of paraffin-embedded tissue slice DNA |
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| CN117737051A true CN117737051A (en) | 2024-03-22 |
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| CN202311809927.5A Pending CN117737051A (en) | 2023-12-27 | 2023-12-27 | Full-automatic extraction method of paraffin-embedded tissue slice DNA |
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- 2023-12-27 CN CN202311809927.5A patent/CN117737051A/en active Pending
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