CN104293772B - The method of FNA material extraction genomic DNA and RNA - Google Patents
The method of FNA material extraction genomic DNA and RNA Download PDFInfo
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Abstract
The invention discloses a kind of method of a fine needle aspiration biopsy material extraction genomic DNA and RNA.The present invention is to mix micro biopsy material to cell pyrolysis liquid and sample with turbine mixer, to be extracted while realization to DNA and RNA, instead of traditional liquid nitrogen grinding or mechanical crushing method due in operating process sample loss it is excessive and DNA and RNA can only be extracted respectively.The operating procedure of the present invention is simple, and cost is low, and required sample size is few, and the experimental implementation time is short, effectively prevents DNA and RNA from degrading；Concentration, purity and the integrality of DNA and RNA after extraction, disclosure satisfy that the analysis requirement of follow-up genomics each side.
Description
Technical field
It is specifically that an a kind of FNA material is carried the present invention relates to separation, the method for preparing or purifying DNA or RNA
Genomic DNA and RNA method are taken, is available for the credit of downstream gene group to analyse.
Background technology
The third time Disease causation sample survey results carried out in China according to the Ministry of Public Health of China and the Department of Science and Technology
Show：Malignant tumour turns into China's second cause of death, accounts for the 22.32% of dead sum, cerebrovascular disease is only second to, in city
City is even more to be in cause of the death first place (account for the dead sum in city 25.0%).Since nearly 30 years, pathogenesis of cancer number is with average annual 3%~5%
Speed increase.At present, traditional imaging diagnosis for medically using, histocytology diagnosis are all to having formed tumor group
The sample for knitting block is analyzed, it is impossible to find the trace of tumour in time.The problem of above-mentioned delayed because traditional method is present, swell
Thus the diagnosis of knurl molecule is born, and its great advantage is that early stage even " extreme early " the distinctive mutator of tumour can be found,
When these gene mutations not yet are accumulated to form tumor tissue, tumour trace can be just found in time, so as to realize effective prevention.Its
In, gene molecule detection is to realize one of the fundamental way of " three is early ".At present, chemotherapy overall efficiency is 30% to 40%, and leads to
Cross genetic test and filter out benefit patient, effective percentage can bring up to 80%.
Tumour Personalized medicine systems biology genome-based technologies comprehensive detection is a kind of brand-new personalized treatment method,
Its principle is to utilize fine needle aspiration biopsy material, using several genes group technology（Lower generation sequencing, rna expression spectrum, copy number become
Change）With system biological method, what is captured is not only the cell aberration of individual, also has answering in all known cell biologicals
Hybridization is mutual.Current research shows that the inducement of cancer is diversified.Tumour Personalized medicine systems biology genome skill
Art comprehensive detection can use different targeted therapies under the conditions of full-length genome monitoring to different people.The project can
The disease applied to healthy population and the analysis of the cancer risk of people at highest risk and cancer patient is visited source, treatment auxiliary and examined simultaneously
Disconnected, medication guide etc..
Tumour Personalized medicine systems biology genome-based technologies comprehensive detection needs to use tumor tissues（Aspiration biopsy or hand
Art is drawn materials）To carry out DNA and RNA separation and Extractions.But total serum IgE preparation method traditional at present（Trizol methods, CTAB-LiCl
Method, CTAB- isopropanol methods, the hot phynol method of guanidinium isothiocyanate）With DNA preparation methods（Strong salty method, anionic detergent method, phenol
Extraction process, water extraction process）Can not fully it meet to the micro biopsy material of a FNA while extracting wanting for DNA and RNA
Ask, Minimal Invasive Biopsy limited material is the current maximum bottleneck for carrying out stage construction full-length genome detection.
The content of the invention
The present invention be exactly in order to solve biopsy material it is few and can not meet the credit of stage construction full-length genome analysis demand the problem of,
And a kind of fine needle aspiration biopsy material extraction genomic DNA and RNA method provided.
The present invention is realized by following technical scheme.
A kind of method of one-time puncture material extraction genomic DNA and RNA, step is as follows：
A. a FNA material is placed in cell pyrolysis liquid, be incubated at room temperature；
B. vortex mixing is carried out to said mixture using turbine mixer, stood, repeating to be vortexed mixes and stand step；
C. the cell pyrolysis liquid mixture of 1/4 volume is taken to be used to separate RNA；
D. remaining cell cracking liquid mixture is used to separate DNA；
It is the step of separation RNA in the step c：
I, adds TRIzol into cell pyrolysis liquid mixture, and homogenized is made with turbine mixer, is stored at room temperature, then adds
Enter chloroform, vibrate, be stored at room temperature；
Centrifuged under II, cryogenic conditions, draw upper strata aqueous phase, added isopropanol precipitating RNA, be stored at room temperature；
Centrifuged under III, cryogenic conditions, discard supernatant, add 75% ethanol washing RNA precipitate；
IV, is centrifuged under the conditions of low temperature, low centrifugal force, is discarded supernatant, is stored at room temperature, and adds the water without RNase, 0.5%
Dodecyl sodium sulfate or 100% deionized formamide solution, suspend precipitation, fully dissolves RNA, -70 DEG C save backup；
It is the step of separation DNA in the step d：
I, adds Proteinase K into residue cracking liquid mixture, mixes, water-bath, vibrates, centrifugation, Aspirate supernatant；
II, adds the isopropanol of 2 times of volumes of supernatant described in I step, mixes, adds after being mixed with supernatant, isopropanol
The equal phenol of volume:Chloroform:Iso pentane alcohol mixture, is mixed, centrifugation；
III, takes upper solution and addition and the isometric chloroform of upper solution:Isoamyl mixed alkoxide solution, is mixed, centrifugation；
IV, takes upper solution and adds the acetic acid ammonia of the volume of upper solution 1/2, adds the nothing of 2 times of volumes of upper solution
Water-ethanol, mixes, is stored at room temperature, and centrifuges；
V, discards supernatant, and precipitation is washed with 70% ethanol, centrifugation；
VI, discards supernatant and tube wall raffinate, adds TE solution and dissolves sediment again, is placed in 4 DEG C or -20 DEG C preservations standby
With.
The present invention so designed, about several milligrams of the biopsy samples obtained to a FNA with turbine mixer and
Cell pyrolysis liquid carries out the traditional liquid nitrogen grinding of vortex mixing replacement or mechanical crushing tissue homogenization process is handled sample, makes
The method operating procedure that DNA and RNA must be extracted is simple, and cost is low；The biopsy material obtained using a FNA can be same
When DNA and RNA are extracted, required sample size is few, reduces the experimental implementation time, effectively prevents DNA and RNA from degrading；
Meanwhile, DNA and RNA concentration, purity and the integrality extracted using method of the present invention disclosure satisfy that follow-up to full base
Because of the requirement of the analyses such as a group copy number, full-length genome expression and full extron sequencing.
Brief description of the drawings
Fig. 1 is the RNA integrity detection figures that sample 1 is extracted；
Fig. 2 is the RNA integrity detection figures that sample 2 is extracted；
Fig. 3 is the RNA integrity detection figures that sample 3 is extracted；
Fig. 4 is the RNA integrity detection figures that sample 4 is extracted；
Fig. 5 is the RNA integrity detection figures that sample 5 is extracted；
Fig. 6 is the DNA integrity detection figures that agarose electrophoresis detects five sample extractions；
Fig. 7 is the RNA full-length genome gene expression analysis figures that sample 1 is extracted；
Fig. 8 is the RNA full-length genome gene expression analysis figures that sample 2 is extracted；
Fig. 9 is the RNA full-length genome gene expression analysis figures that sample 3 is extracted；
Figure 10 is the RNA full-length genome gene expression analysis figures that sample 4 is extracted；
Figure 11 is the RNA full-length genome gene expression analysis figures that sample 5 is extracted；
Figure 12 is the DNA full-length genome copy number analysis of variance figures that sample 1 is extracted;
Figure 13 is the DNA full-length genome copy number analysis of variance figures that sample 2 is extracted;
Figure 14 is the DNA full-length genome copy number analysis of variance figures that sample 3 is extracted;
Figure 15 is the DNA full-length genome copy number analysis of variance figures that sample 4 is extracted;
Figure 16 is the DNA full-length genome copy number analysis of variance figures that sample 5 is extracted.
Embodiment
With reference to embodiment and accompanying drawing, the present invention will be described in detail.
One, main materials and equipment
Sample of the present invention is by a fine needle（7#）Puncture obtained biopsy material.
TRIzol-Invitrogen companies.
The composition of cell pyrolysis liquid is：Trishydroxymethylaminomethane（Tris）Final concentration of 100 mmol/L, ethylenediamine tetraacetic
Acetic acid（EDTA）Final concentration of 500 mmol/L, sodium chloride（NaCl）Final concentration of 20 mmol/L, lauryl sodium sulfate
（SDS）Final concentration of 10%, the final concentration of 20ug/ml of Pancreatic RNase.
The composition of TE solution is：Trishydroxymethylaminomethane（Tris）Final concentration of 10 mmol/L, ethylenediamine tetra-acetic acid
（EDTA）Final concentration of 1 mmol/L.
Phenol:Chloroform:The volume ratio of isoamyl mixed alkoxide solution is 25:24:1.
Chloroform:The volume ratio of isoamyl mixed alkoxide solution is 24:1.
Turbine mixer-Bio Rad Laboratories BR-2000.
Centrifuge-Ai Bende companies of Germany 5804R.
Two .DNA and RNA extraction method
Using the method for a fine needle aspiration biopsy material extraction genomic DNA and RNA, step is as follows：
A. fine needle aspiration biopsy material is all placed in 150~300 ul cell pyrolysis liquids, incubation at room temperature 5~
18min；
B. said mixture be vortexed using turbine mixer and mix 1~3 min, stand 1~5min, this operation weight
It is multiple 2~6 times；
C. the cell pyrolysis liquid mixture of 1/4 volume is taken to be used to separate RNA；
D. remaining cell cracking liquid mixture is used to separate DNA；
It is the step of separation RNA in the step c：
I, adds 0.5~2 ml TRIzol into 50ul cell pyrolysis liquid mixtures, is carried out with turbine mixer at homogenate
Manage 1min；
Homogenised sample is placed 2~8min by II, at room temperature, is kept completely separate nucleic acid-protein compound；
III, adds 0.1~0.4 ml chloroform into homogenised sample, acutely vibrates 10~22s, and room temperature places 2~6min；
8500~12000 × g centrifuges 9~30min under conditions of IV .2~8 DEG C, draws the liquid in upper strata aqueous phase；
V, is transferred to the liquid in aqueous phase in new centrifuge tube, in the isopropanol precipitating aqueous phase for adding 0.2~0.8 ml
RNA, room temperature places 7~20min；
8500~12000 × g centrifuges 9~30min under conditions of VI .2~8 DEG C, discards supernatant；
VII, at least adds the ethanol of 1ml 75% washing RNA precipitate；At 2~8 DEG C, centrifugal force is no more than 7500 × g condition
3~10min of lower centrifugation, discards supernatant；
VIII, room temperatures place 5~10min, add 10~30 water of the μ l without RNase, 0.5% SDS or 100% deionization first
Amide solution, is inhaled with pipettor and beaten several times, and placing 7~20min for 55~60 DEG C dissolves RNA, and -70 DEG C save backup；
It is the step of separation DNA in the step d：
I, takes remaining cracking liquid mixture to be transferred in centrifuge tube, adds the μ l of 500 μ g/ml Proteinase Ks 15~25, mixes；
25~55min of water-bath in 60~70 DEG C of thermostat water baths, or 37 DEG C of 12~24h of water-bath, intermittent oscillation centrifuge tube 3~5 times,
13400 × g centrifuges 3~9min, and Aspirate supernatant is into new centrifuge tube；
II, adds 340 μ l isopropanol, reverses and mixes；
III, adds 510 μ l phenol:Chloroform:The vibration of isoamyl mixed alkoxide solution is mixed, and 13400 × g centrifuges 3~9min；
IV, takes upper solution to a new centrifuge tube, adds 500 μ l chloroform:Isoamyl mixed alkoxide solution, vibration is mixed,
13400 × g centrifuges 2~8min；
V, takes upper solution to a new centrifuge tube, adds 250 μ l 7.5mol/L acetic acid ammonia, adds the anhydrous of 1ml
Ethanol, is mixed, and precipitation at room temperature 1~6min, 13400 × g centrifuge 6~15min；
VI, discards supernatant, removes the residual liquid on tube wall；
VII, 1~2 ml, 70% ethanol washing precipitates 1~3 time, 13400 × g centrifuges 3~9min；
VIII, discards supernatant, removes the residual liquid on tube wall；
Ⅸ, adds 25~40 μ l TE solution dissolve sediment again, be placed in 4 DEG C or -20 DEG C save backup.
Three, results
1. the RNA pattern detections extracted
（1）RNA concentration of specimens and purity detecting
The RNA total amounts separated using the method for the invention between 140~420 ng, concentration 10~30 ng/ul it
Between, the ratios of OD 260/280 are between 1.8~2.2（NanoDrop 8000, Thermo Scientific companies）, referring to table
1。
（2）RNA sample integrities are detected
The integrality of the 5 RNA samples extracted is 8~10（RIN Number）Between（Agilent2100,
Agilent companies）, referring to accompanying drawing 1~5.
2. the DNA sample detection extracted
（1）DNA sample concentration and purity detecting
The DNA total amounts extracted using method of the present invention are between 600~1200ng, and concentration is in 20~40ng/ul
Between, OD260/280 ratios are between 1.8~2.2（NanoDrop 8000, Thermo Scientific companies）, it is shown in Table
2。
（2）DNA sample integrity detection
The DNA integralities of 5 samples are detected using 1% agarose electrophoresis, referring to accompanying drawing 6, as a result show that genomic DNA does not have
Degrade.
3. with separation RNA samples and Affymetrix Human U133plus2.0（Affymetrix companies）Chip enters
Row full-length genome gene expression analysis
To prove separated RNA quality, full-length genome is carried out with Affymetrix Human U133plus2.0 chips
Gene expression analysis, detects its hybridization signal and quality, referring to Fig. 7~11.As a result show：The hybridization signal of 5 samples all exists
More than 200, have no hybrid context；The scale factor of 5 samples（SF）All below 3, the percentage of expressing gene is left 50%
The right side, quality complies fully with requirement, referring to table 3.
4. with separation DNA sample and Affymetrix CytoScanHD（Affymetrix companies）Chip carries out full genome
Group copy number analysis of variance
To prove that gained DNA meets the standard of downstream analysis, with separation DNA and Affymetrix CytoScanHD chips
Full-length genome copy number analysis of variance is carried out, referring to Figure 12~16, is as a result shown：5 sample indices reach chip matter
Control index：1. middle position definitely matches difference (Mapd)≤0.25；2. SNP (SNP) >=15；3. fluctuation
（Wavines）≤0.12.
Can the quick, letter from several milligrams of the biopsy material that a FNA is obtained using method of the present invention
Just separate enough DNA and RNA and carry out the analysis of full-length genome copy number, full-length genome expression and full extron sequencing.This hair
Bright to solve current biopsy material few and can not meet the contradiction between stage construction full-length genome credit analysis demand, is individuation doctor
The genetic test for the treatment of provides new approach.
Claims (2)
1. a kind of method of a FNA material extraction genomic DNA and RNA, step is as follows：
A. a FNA material is placed in by the mmol/L of trishydroxymethylaminomethane 100, ethylenediamine tetra-acetic acid 500
In mmol/L, the mmol/L of sodium chloride 20, lauryl sodium sulfate 10%, the cell pyrolysis liquid of the μ g/ml of Pancreatic RNase 20 compositions,
It is incubated at room temperature 5~18min；
B. said mixture be vortexed using turbine mixer and mix 1~3min, stand 1~5min, repeating to be vortexed mixes
With standing step 2~6 time；
C. the cell pyrolysis liquid mixture of 1/4 volume is taken to be used to separate RNA；
D. remaining cell cracking liquid mixture is used to separate DNA；
It is the step of separation RNA in the step c：
I, adds TRIzol into cell pyrolysis liquid mixture, and homogenized is made with turbine mixer, is stored at room temperature 2~8min,
Chloroform is added, 10~22s is vibrated, is stored at room temperature 2~6min；
8500~12000 × g centrifuges 9~30min under II, cryogenic conditions, draws upper strata aqueous phase, adds isopropanol precipitating RNA, room
Temperature stands 7~20min；
8500~12000 × g centrifuges 9~30min under III, cryogenic conditions, discards supernatant, adds 75% ethanol washing RNA and sinks
Form sediment；
IV, centrifuges 3~10min under conditions of low temperature, low centrifugal force, discards supernatant, is stored at room temperature 5~10min, adds nothing
RNase water, 0.5% dodecyl sodium sulfate or 100% deionized formamide solution, suspend precipitation, fully dissolves RNA, -70
DEG C save backup；
It is the step of separation DNA in the step d：
I, to residue cracking liquid mixture in add Proteinase K, mix, in 60~70 DEG C of thermostat water baths water-bath 25~
55min, or 37 DEG C of 12~24h of water-bath, intermittent oscillation 3~5 times, 13400 × g centrifuge 3~9min, Aspirate supernatant；
II, adds the isopropanol of 2 times of volumes of supernatant described in the steps of d I, mixes, adds body after being mixed with supernatant, isopropanol
The equal phenol of product:Chloroform:Isoamyl mixed alkoxide solution, is mixed, centrifugation；
III, takes upper solution and addition and the isometric chloroform of upper solution:Isoamyl mixed alkoxide solution, is mixed, centrifugation；
IV, takes upper solution and adds the ammonium acetate of the volume of upper solution 1/2, adds the anhydrous second of 2 times of volumes of upper solution
Alcohol, mixes, is stored at room temperature, and centrifuges；
V, discards supernatant, and precipitation is washed with 70% ethanol, centrifugation；
VI, discards supernatant and tube wall raffinate, adds TE solution and dissolves sediment again, is placed in 4 DEG C or -20 DEG C save backup.
2. the method for a FNA material extraction genomic DNA according to claim 1 and RNA, it is characterised in that：
The step c, centrifugal force is no more than 7500 × g in IV.
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| CN105368816B (en) * | 2015-11-25 | 2019-01-08 | 湖北省农业科学院畜牧兽医研究所 | Method for separating DNA, RNA and protein from single plant sample |
| CN105385680B (en) * | 2015-12-24 | 2019-01-25 | 天津脉络生物科技有限公司 | For the reagent that DNA and RNA is extracted simultaneously, extracting method and purposes |
| CN112195176B (en) * | 2020-09-30 | 2023-05-02 | 天津大潮基因科技有限公司 | Method for separating and purifying nucleic acid solid from biological material |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680295A (en) * | 2012-05-21 | 2012-09-19 | 江苏省中医院 | Method for extracting residual nucleic acid from HE (haematoxylin eosin) dyeing piece |
| CN103224929A (en) * | 2013-04-09 | 2013-07-31 | 南京紫霄科技有限公司 | Method for rapidly concentrating and extracting nucleic acid target cells from sample |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680295A (en) * | 2012-05-21 | 2012-09-19 | 江苏省中医院 | Method for extracting residual nucleic acid from HE (haematoxylin eosin) dyeing piece |
| CN103224929A (en) * | 2013-04-09 | 2013-07-31 | 南京紫霄科技有限公司 | Method for rapidly concentrating and extracting nucleic acid target cells from sample |
Non-Patent Citations (2)
| Title |
|---|
| 一种高效同时提取微量组织中总RNA、DNA和蛋白的技术方法的改进;汪军等;《肝胆胰外科杂志》;20120731;第24卷(第4期);第309页左栏第2段，第309页左栏倒数第1段-右栏第2段，第313页右栏第1段，图1 * |
| 改良TRIzol法同步提取血液RNA和DNA;秦娟娟等;《法医学杂志》;20130630;第29卷(第3期);209-211 * |
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