CN109371107A - A kind of rapid automatized extracting method and reagent of paraffin section tissue nucleic acid - Google Patents
A kind of rapid automatized extracting method and reagent of paraffin section tissue nucleic acid Download PDFInfo
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- CN109371107A CN109371107A CN201811585769.9A CN201811585769A CN109371107A CN 109371107 A CN109371107 A CN 109371107A CN 201811585769 A CN201811585769 A CN 201811585769A CN 109371107 A CN109371107 A CN 109371107A
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Abstract
The invention discloses a kind of rapid automatized extracting method of paraffin section tissue nucleic acid and reagent, the reagent includes dewaxing agent, protein enzyme solution, lysate, cleaning solution A, cleaning solution B and eluent；Its method is to use nontoxic dewaxing agent fast dewaxing, after ethanol washing, cracking is added containing the protease of SDS and urea and is digested to liquid, it is transferred in automation magnetic bead extraction apparatus and extracts, successively carry out cracking and magnetic bead absorption, washing A, washing B, eluent, runing time in about 30 minutes machines, the extraction of achievable 32 parts or even 96 parts samples；The rapid automatized extracting method and reagent of paraffin section tissue nucleic acid of the invention, after completing conventional first time protease and being incubated for, to be incubated for dissolution system be warming up to after high temperature maintain 3 ~ take out after five minutes, it is cooled to room temperature to it, protease is added into the reaction system again, being incubated for again 10 ~ 30 minutes can make tissue be completely dissolved disappearance, and without obvious particulate matter, solution is limpid.
Description
Technical field
The present invention relates to a kind of rapid automatized extracting method of paraffin section tissue nucleic acid and reagents, belong to biological medicine
Technical field.
Background technique
Oncogene detection is current popular clinical molecular diagnosis field, passes through the separation to tumor tissues DNA, RNA
Purifying, carry out detection of nucleic acids understand tumour characterization of molecules, thus predict, diagnosing tumour so judge patient's prognosis, recurrence give birth to
It deposits, curative effect of medication etc.；Paraffin section investing tissue is the most common oncogene detection sample, because paraffin embedding is that room temperature is long
Phase saves the classical way of histone and nucleic acid molecules, and nearly all medical institutions can be saved swollen using paraffin embedding method
Tumor tissue；During paraffin embedding, flesh tissue is fixed by organic solvent and is gradually dehydrated, and the ingredient of water is by stone in tissue
Wax oil substitution, so that biomolecule be made completely to be saved；And during paraffin-embedded tissue extracts DNA and RNA, it is necessary to
By the paraffin component dissolution in tissue, being substituted by water, become dewaxing, the tissue of dewaxing generally uses protease to be digested,
With substances such as released dna, RNA；Dewaxing and course of dissolution are generally completed by dimethylbenzene and Proteinase K
General paraffin section organizational requirements thickness between 5 ~ 6 microns, thin slice be conducive to the infiltration of dimethylbenzene equal solvent with
The digestion of protease, thus keep extraction more thorough, but in actual operation, the thickness of paraffin section is in different medical mechanism
And different medical mechanism and different sectioning persons have biggish difference when cutting；It is biggish especially to carry out nucleic acid dosage
When genetic test such as 21 quantitative gene expression of breast cancer, tester, which may require that, cuts larger amount of paraffin organization, and paraffin is cut at this time
Piece quantity and slice thickness may dramatically increase；Detection operator can be molten using the method for extending protease incubation time at this time
Solving larger amount of paraffin organization, protease digestion incubation time is 15 minutes to 3 hours when general paraffin organization nucleic acid extraction, but
For being sliced thicker and a fairly large number of paraffin section, it can not thoroughly be dissolved extending the time to 24 hours, container
It bottom still can residual solids tissue；Incubation time is too long to make partial nucleic acid substance occur degrading and be broken, and organize remnants'
In the presence of nucleic acid extraction amount can not only reduced, but also it can make to have dissolved digestion the extracted nucleic acid of portion of tissue to the generation entirely organized
There is deviation in table, to influence measurement result.
Summary of the invention
To solve the above problems, the invention proposes a kind of rapid automatized extracting method of paraffin section tissue nucleic acid with
Reagent, provides a kind of convenient and efficient, using safe and non-toxic reagent, and is operated.
The reagent of paraffin section tissue nucleic acid of the invention, including dewaxing agent, protein enzyme solution, lysate, cleaning solution A,
Cleaning solution B and eluent；
The dewaxing agent is D- limonene；
The protein enzyme solution is by 5 ~ 20mg/ml Proteinase K, 0.1 ~ 0.8%SDS, 0.2 ~ 9M urea, 5 ~ 200m M Tris-
HCl, 0.1 ~ 10m M EDTA.Na2 composition；
The lysate by 4.5M guanidine thiocyanate, 0.9%DTT, 5% polidocanol, 50m M sodium citrate buffer solution, 4M urea and
0.5%SDS composition；
The cleaning solution A is made of 2M guanidine thiocyanate, 30% ethyl alcohol, 0.25%SDS and 2M urea；
The cleaning solution B is made of 80% ethyl alcohol, 20m M sodium chloride, 2m MTris and 0.1%Tween20；
The eluent is made of 10m M Tris-HCL and 0.2m M EDTA.
Further, the protein enzyme solution is by 20mg/ml Proteinase K, 0.2%SDS, 6M urea, 20m M Tris-
HCl, 2m M EDTA.Na2,5Mm calcium chloride and preservative composition.
Further, the cleaning solution A is made of 2M guanidine thiocyanate, 30% ethyl alcohol, 0.25%SDS, 2M urea and pigment, and
Its pH value of cleaning solution A is 7.5.
Still further, the pigment is phenol red.
Further, the eluent is by 10m M Tris-HCL, 0.2m M EDTA.Na2 and 0.2% P-hydroxybenzoic acid
Methyl esters composition.
Further, its pH value of the Tris-HCl is 8.0.
A kind of rapid automatized extracting method of paraffin section tissue nucleic acid, comprising the following steps:
Step 1 cuts the embedding paraffin sample of 85 ~ 10 μ m-thicks into 1.5ml or 2ml centrifuge tube, if embedding paraffin sample
This surface is exposed in air, abandons previous 2 ~ 3；1 ~ 1.2ml D- limonene, the vortex that closes the lid oscillation is added
Room temperature high speed centrifugation 1 ~ 5 minute after 10 seconds；
Step 2 abandons supernatant, and 1 ~ 1.2ml dehydrated alcohol is added, and oscillation mixes, and room temperature high speed centrifugation 1 ~ 5 minute, moves on abandoning
Clearly, lid is opened, is placed at room temperature for or 37 DEG C of placements 10min, until the volatilization of remaining ethyl alcohol is completely；
Step 3, tissue digestion: being added 200 μ l protein enzyme solutions in the dry tissue of dewaxing, and oscillation mixes, and 70 DEG C 10 ~ 30
90 DEG C 10 ~ 30 minutes after minute, brief centrifugation remove inside pipe wall droplet；
Then 310 μ l dehydrated alcohols or different are added after 1 ~ 2 mixing is played in suction in step 4, the first row of transfer liquid to deep-well plates
Propyl alcohol, upper magnetic bead extraction apparatus；
Step 5, the cracking of magnetic bead extraction apparatus automatic running, adsorption step, cleaning solution A washing step and cleaning solution B washing step,
Elution is finally heated in eluent；End of run eluent is to be used for subsequent molecular biological analysis containing nucleic acid-templated.
Further, added with 400 μ l lysates and 20 μ l magnetic beads in the first row of the deep-well plates in the step 4.
Further, it is stirred 5 ~ 20 minutes repeatedly in the adsorption step in the step 5 using stirring set；Washing step
Middle to be washed 1 ~ 2 time with cleaning solution A and cleaning solution B respectively, wash time is 1 ~ 2 minute；Its heating of eluent is washed in elution step
De- temperature is 50 ~ 80 DEG C, and elution time is 3 ~ 10 minutes.
The present invention compared with prior art, the rapid automatized extracting method of paraffin section tissue nucleic acid of the invention with
Reagent, provides a kind of convenient and efficient, uses safe and non-toxic reagent；It, will and after completing conventional first time protease and being incubated for
Be incubated for dissolution system be warming up to after high temperature maintain 3 ~ take out after five minutes, be cooled to room temperature to it, then be added into the reaction system
Protease, then being incubated for 10 ~ 30 minutes can make tissue be completely dissolved disappearance, without obvious particulate matter, solution is limpid.
Detailed description of the invention
Fig. 1 is to be utilized respectively QIAGEN paraffin tissue sections nucleic acid extraction kit and paraffin section tissue core of the invention
The rapid automatized extracting method of acid extracts the nucleic acid-templated result schematic diagram for carrying out ultraviolet determination.
Specific embodiment
The reagent of paraffin section tissue nucleic acid of the invention, including dewaxing agent, protein enzyme solution, lysate, cleaning solution A,
Cleaning solution B and eluent；
The dewaxing agent is D- limonene；
The protein enzyme solution is by 5 ~ 20mg/ml Proteinase K, 0.1 ~ 0.8%SDS, 0.2 ~ 9M urea, 5 ~ 200m M Tris-
HCl, 0.1 ~ 10m M EDTA.Na2 composition；
The lysate by 4.5M guanidine thiocyanate, 0.9%DTT, 5% polidocanol, 50m M sodium citrate buffer solution, 4M urea and
0.5%SDS composition；
The cleaning solution A is made of 2M guanidine thiocyanate, 30% ethyl alcohol, 0.25%SDS and 2M urea；
The cleaning solution B is made of 80% ethyl alcohol, 20m M sodium chloride, 2m MTris and 0.1%Tween20；
The eluent is made of 10m M Tris-HCL and 0.2m M EDTA.
The protein enzyme solution is by 20mg/ml Proteinase K, 0.2%SDS, 6M urea, 20m M Tris-HCl, 2m M
EDTA.Na2,5Mm calcium chloride and preservative composition, and protein enzyme solution is filtered through 0.45 μm of filter membrane to remove miscellaneous bacteria.
The cleaning solution A utilizes Tris by 2M guanidine thiocyanate, 30% ethyl alcohol, 0.25%SDS, 2M urea and pigment composition
Alkali and hydrochloric acid adjust its pH value of cleaning solution A to 7.5.
The pigment is phenol red.
The eluent is made of 10m M Tris-HCL, 0.2m M EDTA.Na2 and 0.2% methyl p-hydroxybenzoate.
Its pH value of the Tris-HCl is 8.0.
The rapid automatized extracting method of paraffin section tissue nucleic acid of the invention, comprising the following steps:
Step 1 cuts the embedding paraffin sample of 85 ~ 10 μ m-thicks into 1.5ml centrifuge tube, if embedding paraffin sample surface
It is exposed in air, abandons previous 2 ~ 3；1 ~ 1.2ml D- limonene is added, after the vortex that closes the lid vibrates 10 seconds
Room temperature high speed centrifugation 1 ~ 5 minute；
Step 2 abandons supernatant, and 1 ~ 1.2ml dehydrated alcohol is added, and oscillation mixes, and room temperature high speed centrifugation 1 ~ 5 minute, moves on abandoning
Clearly, lid is opened, 10min is placed at room temperature for, until the volatilization of remaining ethyl alcohol is complete；
Step 3, tissue digestion: being added 200 μ l protein enzyme solutions in the dry tissue of dewaxing, and oscillation mixes, and 70 DEG C 10 ~ 30
90 DEG C 10 ~ 30 minutes after minute, brief centrifugation remove inside pipe wall droplet；
Step 4, the first row of transfer liquid to deep-well plates, wherein suction is made a call to 1 ~ 2 time added with 400 μ l lysates and 20 μ l magnetic beads
After mixing, 310 μ l dehydrated alcohols or isopropanol, upper magnetic bead extraction apparatus is then added；
Step 5, the cracking of magnetic bead extraction apparatus automatic running, adsorption step (being stirred 15 minutes repeatedly using stirring set), cleaning solution A
(1 minute) washing step and cleaning solution B(1 minutes) washing step, finally heats elution, wherein utilize washing in eluent
Liquid A is washed 1 time, is washed 2 times using cleaning solution B, is eluted 5 minutes at a temperature of 80 DEG C；End of run eluent is to contain nucleic acid
Template is used for subsequent molecular biological analysis.
It is nucleic acid-templated using the extraction of QIAGEN paraffin tissue sections nucleic acid extraction kit,
Paraffin-embedded tissue is cut into 1.5 ~ 2ml centrifuge tube, does not exceed 25mg by step 1；Dimethylbenzene 1.2ml is added,
Acutely oscillation, most high speed room temperature are centrifuged 5 min, move and abandon supernatant；
1.2ml dehydrated alcohol is added in step 2, and oscillation mixes, and most high speed room temperature is centrifuged 5min, moves and abandons supernatant, and repeats this step
Rapid 1 ~ 2 time；
Step 3 opens lid, 37 DEG C of 10 ~ 15min of placement, until ethyl alcohol volatilization is complete；
180 μ l buffer ATL are added in step 4, so that precipitating is sufficiently hanged；
Step 5, be added 20 μ l Proteinase Ks, after mixing 56 DEG C be incubated for organize completely cracking (general 1 ~ 3 hour, individual 6 ~ 8
Hour, usually it is incubated overnight more convenient), cracking process can be accelerated by mixing when being incubated for；
Step 6 takes out incubation tube, mixes 1 ~ 15 second, and 200 μ l buffer ATL are added, and 200 μ of dehydrated alcohol is added after mixing
Liquid (including any sediment) is added on nucleic acid absorption column by l, mixing；6000g x 1min discards 2ml casing, transfer
Pillar is into a new 2ml casing；
500 μ l Buffer AW1,6000g x 1min are added in step 7, discard 2ml casing, transfer pillar to a new 2ml
In casing；
500 μ l Buffer AW2,6000g x 1min are added in step 8, discard 2ml casing, transfer pillar to a new 2ml
In casing；
Step 9,2 min of 20000g x discard 2ml casing, shift pillar into a new 1.5ml collecting pipe；
Step 10 is vertically added 100 μ l of eluent in the overcentre of film, is stored at room temperature 1 minute, infiltrates film sufficiently；
Step 11,6000g x 1min, the liquid in collecting pipe is as nucleic acid-templated, is used for subsequent molecular biological
Analysis.
It is utilized respectively QIAGEN paraffin tissue sections nucleic acid extraction kit and paraffin section tissue nucleic acid of the invention
Rapid automatized extracting method extraction is nucleic acid-templated, and will be extracted using QIAGEN paraffin tissue sections nucleic acid extraction kit
Sample is denoted as S1 and S2, and the sample that the rapid automatized extracting method of paraffin section tissue nucleic acid of the invention takes is denoted as S11
And S22, wherein S1 and S11, S2 and S22 are with a paraffin tissue sections sample；S1, S2, S11 and S22 are carried out respectively purple
Outer measurement, as shown in Figure 1, having the advantages that significant, of the invention paraffin section tissue nucleic acid by the visible present invention of upper result
Rapid automatized extracting method can automate, is high-throughput, extract yield height, A260/280 is between 1.8 ~ 2.0, A260/
For A230 between 1.5 ~ 2.5, purity is good.
The rapid automatized extracting method and reagent of paraffin section tissue nucleic acid of the invention, provide it is a kind of convenient and efficient,
Use safe and non-toxic reagent；And (general incubation time is 60 ~ 120 points after completing conventional first time protease and being incubated for
Clock), will be incubated for dissolution system be warming up to high temperature (between 80 ~ 90 degree) maintain 3 afterwards ~ take out after five minutes, be cooled to room temperature to it,
Protease is added into the reaction system again, then being incubated for 10 ~ 30 minutes can make tissue be completely dissolved disappearance, without obvious particle
Object, solution are limpid.
Above-described embodiment is only better embodiment of the invention, therefore all according to structure described in present patent application range
It makes, the equivalent change or modification that feature and principle are done, is included in the scope of the patent application of the present invention.
Claims (9)
1. a kind of reagent of paraffin section tissue nucleic acid, which is characterized in that including dewaxing agent, protein enzyme solution, lysate, washing
Liquid A, cleaning solution B and eluent；
The dewaxing agent is D- limonene；
The protein enzyme solution is by 5 ~ 20mg/ml Proteinase K, 0.1 ~ 0.8%SDS, 0.2 ~ 9M urea, 5 ~ 200m M Tris-
HCl, 0.1 ~ 10m M EDTA.Na2 composition；
The lysate by 4.5M guanidine thiocyanate, 0.9%DTT, 5% polidocanol, 50m M sodium citrate buffer solution, 4M urea and
0.5%SDS composition；
The cleaning solution A is made of 2M guanidine thiocyanate, 30% ethyl alcohol, 0.25%SDS and 2M urea；
The cleaning solution B is made of 80% ethyl alcohol, 20m M sodium chloride, 2m MTris and 0.1%Tween20；
The eluent is made of 10m M Tris-HCL and 0.2m M EDTA.
2. the reagent of paraffin section tissue nucleic acid according to claim 1, which is characterized in that the protein enzyme solution by
20mg/ml Proteinase K, 0.2%SDS, 6M urea, 20m M Tris-HCl, 2m M EDTA.Na2,5Mm calcium chloride and preservative
Composition.
3. the reagent of paraffin section tissue nucleic acid according to claim 1, which is characterized in that the cleaning solution A is by 2M sulphur
Cyanic acid guanidine, 30% ethyl alcohol, 0.25%SDS, 2M urea and pigment composition, and its pH value of cleaning solution A is 7.5.
4. the reagent of paraffin section tissue nucleic acid according to claim 3, which is characterized in that the pigment is phenol red.
5. the reagent of paraffin section tissue nucleic acid according to claim 1, which is characterized in that the eluent is by 10m M
Tris-HCL, 0.2m M EDTA.Na2 and 0.2% methyl p-hydroxybenzoate composition.
6. the reagent of paraffin section tissue nucleic acid according to claim 1, which is characterized in that its PH of the Tris-HCl
Value is 8.0.
7. a kind of rapid automatized extracting method of paraffin section tissue nucleic acid, which comprises the following steps:
Step 1 cuts the embedding paraffin sample of 85 ~ 10 μ m-thicks into 1.5ml or 2ml centrifuge tube, if embedding paraffin sample
This surface is exposed in air, abandons previous 2 ~ 3；1 ~ 1.2ml D- limonene, the vortex that closes the lid oscillation is added
Room temperature high speed centrifugation 1 ~ 5 minute after 10 seconds；
Step 2 abandons supernatant, and 1 ~ 1.2ml dehydrated alcohol is added, and oscillation mixes, and room temperature high speed centrifugation 1 ~ 5 minute, moves on abandoning
Clearly, lid is opened, is placed at room temperature for or 37 DEG C of placements 10min, until the volatilization of remaining ethyl alcohol is completely；
Step 3, tissue digestion: being added 200 μ l protein enzyme solutions in the dry tissue of dewaxing, and oscillation mixes, and 70 DEG C 10 ~ 30
90 DEG C 10 ~ 30 minutes after minute, brief centrifugation remove inside pipe wall droplet；
Then 310 μ l dehydrated alcohols or different are added after 1 ~ 2 mixing is played in suction in step 4, the first row of transfer liquid to deep-well plates
Propyl alcohol, upper magnetic bead extraction apparatus；
Step 5, the cracking of magnetic bead extraction apparatus automatic running, adsorption step, cleaning solution A washing step and cleaning solution B washing step,
Elution is finally heated in eluent；End of run eluent is to be used for subsequent molecular biological analysis containing nucleic acid-templated.
8. the rapid automatized extracting method of paraffin section tissue nucleic acid according to claim 7, which is characterized in that described
Added with 400 μ l lysates and 20 μ l magnetic beads in the first row of deep-well plates in step 4.
9. the rapid automatized extracting method of paraffin section tissue nucleic acid according to claim 7, which is characterized in that described
It is stirred 5 ~ 20 minutes repeatedly in adsorption step in step 5 using stirring set；Respectively with cleaning solution A and washing in washing step
Liquid B is washed 1 ~ 2 time, and wash time is 1 ~ 2 minute；In elution step eluent its to heat eluting temperature be 50 ~ 80 DEG C, when elution
Between be 3 ~ 10 minutes.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109957563A (en) * | 2019-05-05 | 2019-07-02 | 上海睿璟生物科技有限公司 | DNA extraction method in paramagnetic particle method FFPE tissue |
| CN110093342A (en) * | 2019-03-28 | 2019-08-06 | 凡知医疗科技(江苏)有限公司 | A method of it is sliced from paraffin-embedded tissue and extracts nucleic acid |
| CN110835628A (en) * | 2019-11-25 | 2020-02-25 | 宁波艾捷康宁生物科技有限公司 | Paraffin removal lysate for extracting genome DNA of paraffin section, extraction kit and extraction method |
| CN111172239A (en) * | 2020-02-28 | 2020-05-19 | 上海思路迪医学检验所有限公司 | Virus sample preserving fluid, nucleic acid extraction reagent and virus nucleic acid extraction method |
| 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 |
| 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 |
| CN114891778A (en) * | 2022-03-31 | 2022-08-12 | 杭州丹威生物科技有限公司 | Method and kit for full-automatic extraction of DNA of paraffin-embedded tissue section |
| CN117821448A (en) * | 2024-03-05 | 2024-04-05 | 苏州麦锐克生物科技有限公司 | Serum/plasma miRNA full-automatic extraction kit and extraction method thereof |
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| CN114891778A (en) * | 2022-03-31 | 2022-08-12 | 杭州丹威生物科技有限公司 | Method and kit for full-automatic extraction of DNA of paraffin-embedded tissue section |
| CN117821448A (en) * | 2024-03-05 | 2024-04-05 | 苏州麦锐克生物科技有限公司 | Serum/plasma miRNA full-automatic extraction kit and extraction method thereof |
| CN117821448B (en) * | 2024-03-05 | 2024-05-17 | 苏州麦锐克生物科技有限公司 | Serum/plasma miRNA full-automatic extraction kit and extraction method thereof |
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