CN102807975A - Method for rapidly extracting nucleic acid from biological sample - Google Patents
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- CN102807975A CN102807975A CN2011101432418A CN201110143241A CN102807975A CN 102807975 A CN102807975 A CN 102807975A CN 2011101432418 A CN2011101432418 A CN 2011101432418A CN 201110143241 A CN201110143241 A CN 201110143241A CN 102807975 A CN102807975 A CN 102807975A
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Abstract
The present invention relates to a method for rapidly extracting nucleic acid from a biological sample. The method comprises: taking a biological sample, placing the sample in an aqueous solution of chelex-100 or a DEPC aqueous solution of chelex-100, and heating for 1-50 minutes at a temperature of 80-100 DEG C; placing an extraction column into a centrifuge tube, adding the resulting liquid to the extraction column, and centrifugating for 2-120 seconds at a rotation speed of 1000-20000 rotation/min; and taking the centrifugated liquid to carry out a PCR reaction or a RT-PCR reaction. According to the present invention, chelex-100 and a high temperature heating method are adopted to extract so as to eliminate a protease hydrolysis step, eliminate a paraffin section dewaxing step, remove an organic solvent xylene, and substantially shorten a time; and a retention membrane is placed in the extraction column so as to remove adverse effects on the PCR reaction or the RT-PCR reaction by the liquid paraffin and the chelex-100, and improve sensitivity of the PCR reaction or the RT-PCR reaction.
Description
Technical field
The present invention relates to a kind of from biological specimen the method for rapid extraction nucleic acid.
Background technology
Dna molecular is the basic material of molecular biology research, can take different method for extracting to obtain the DNA that quality and quantity does not wait according to different experiment purposes.The method of extracting DNA generally has following several kinds at present:
(1) anionic detergent method: make protein denaturation with stain removers such as SDS or xylic acid sodium, can directly from biomaterial, extract DNA.In the cell between DNA and the protein normal electrostatic attraction or the co-ordination bond of borrowing combines because anionic detergent can destroy this valence link,, react but anionic detergent usually influences subsequent P CR so anionic detergent commonly used extracts DNA.
(2) water extraction process: utilize nucleic acid to be dissolved in the character of water, after the histocyte fragmentation, remove RNA with low salts solution, will precipitate soluble in waterly then, DNA fully is dissolved in the water, supernatant is collected in centrifugal back.In supernatant, add solid sodium chloride and be adjusted to 2.6M.Add 2 times of volume 95% ethanol, stir out with paddling process immediately, use the washing of 66%, 80% and 95% ethanol and third bronze medal then respectively, at last at air drying, both got the DNA sample, protein contnt is higher among the DNA that this method is extracted, so generally need not.
(3) phenol extraction process: phenol has suppressed the Degradation of DNase simultaneously as protein denaturant.When handling homogenate with phenol, because albumen and DNA link button are disconnected, the protein molecular surface contains again that a lot of polar groups are similar with phenol to mix.Protein molecular is dissolved in the phenol phase, and DNA is soluble in the aqueous phase.Take out water layer after the centrifugal layering, repeatedly repetitive operation remerges the water that contains DNA, utilizes nucleic acid to be insoluble to the character of alcohol, uses ethanol sedimentation DNA.This moment, DNA was a ten minutes heavy-gravity material, and one of the very long coiled of useable glass takes out.The characteristics of this method are to make the DNA of extraction keep native state.
The phenol extraction process is the most conventional method of extracting DNA, but because will use phenol, harmful to the experimenter, therefore the commercial kit of utilizing the method for adsorbing post to extract DNA is also arranged at present.Because need the Proteinase K treating processes, generally need extractive process through 3-4 hour, could get into subsequent P CR reaction.
There is a large amount of various diseases formaldehyde fixed paraffin-embedded tissues (formalin fixed and paraffin embedded tissues in hospital pathology department; FFPET); For providing great deal of information source, targeted therapy in recent years, molecular pathology research make from paraffin-embedded tissue extracting DNA become the focus of research.Yet the degraded of the wax embedding tissue DNA after the formalin fixed is serious; The file FFPET tissue slice amount of getting is limited; Tradition phenol chloroform extraction DNA step is numerous and diverse; The serious grade of leaching process loss all restricted the Application Research of paraffin organization in molecular pathology, and how efficient, high quality extracts that DNA carries out molecular studies and diagnoses most important utilizing paraffin organization in the micro-paraffin organization.
Tradition extracting DNA in the paraffin organization needs earlier to remove YLENE with absolute ethyl alcohol then with the YLENE dewaxing, and dewaxing process is loaded down with trivial details, needs 3～4 hours at least, and YLENE is organic solvent, and is harmful to experimental implementation person's health.After having taken off wax, sample also needs could get into subsequent P CR reaction again through 3～4 hours extracting.
Chelex-100 is a kind of chelate resin of being made up of vinylbenzene and benzene divinyl interpolymer; Its suspension is under alkaline environment (pH10～11) and 100 ℃ of conditions; Can cause membranolysis and the DNA sex change is discharged; But and the combination polyvalent cation of Chelex-100 highly selective, remove the polyvalent metal ion in sample and the damping fluid, avoid the degraded of template DNA in the boiling part.Chelex-100 extracts DNA and has economy, advantage such as easy, efficient, has been used at present extract DNA from whole blood or blood, seminal fluid or seminal stain, mixed stain, hair, culturing cell etc.But generally also need could get into subsequent P CR reaction through 2～3 hours extractive process.
The report that adopts Chelex-100 to extract the genetic material in the paraffin section has been arranged at present; In article " comparisons of four a kinds of micro-paraffin organization DNA extraction methods " literary composition of delivering on Hainan medical science institute journal in 2010, just adopted Chelex-100 to extract the DNA in the paraffin section, but in the document; Before adopting Chelex-100 to extract; Still adopt the step of YLENE dewaxing, washing with alcohol, thereby made complex steps, operated not enough simple and fast.For this reason, the contriver has proposed a kind of simple and fast, the respond well method of from biological specimen, extracting DNA.
But still do not adopt Chelex-100 to extract the report of RNA at present, for this reason, the contriver has proposed simply quick, the respond well method of from biological specimen, extracting RNA of a kind of Chelex-100 of employing.
Summary of the invention
Goal of the invention of the present invention is to propose a kind of rapid extracting method of nucleic acid.
In order to realize goal of the invention of the present invention, the technical scheme that is adopted is:
The present invention relates to a kind of from biological specimen the method for rapid extraction DNA, described method may further comprise the steps:
1. the biological specimen that takes a morsel, said biological specimen is selected from pathological tissue paraffin section sample, blood cake seminal stain or semen sample, tissue juice sample, CSF sample, blood sample, serum sample, plasma sample, urine specimen, hydrops sample, histocyte sample;
2. biological specimen is placed the aqueous solution of chelex-100, the concentration of the aqueous solution of described chelex-100 is 1～80g/100ml;
3.80 heated 1～50 minute under～100 ℃ of conditions;
4. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 1000～20000 rev/mins, centrifugal 2～120 seconds;
5. collect the liquid under leaving, be used for the PCR reaction.
The invention still further relates to a kind of from biological specimen the method for rapid extraction RNA, it is characterized in that described method may further comprise the steps:
1. the biological specimen that takes a morsel, said biological specimen is selected from pathological tissue paraffin section sample, blood cake seminal stain or semen sample, tissue juice sample, CSF sample, blood sample, serum sample, plasma sample, urine specimen, hydrops sample, sample of hair, histocyte sample;
2. place the DEPC aqueous solution of 1～80%chelex-100;
3.80 heated 1～50 minute under～100 ℃ of conditions;
4. the extracting column jecket is put into centrifuge tube, the liquid that obtains in the step (3) is joined in the extracting column jecket, 1000～20000 rev/mins, centrifugal 2～120 seconds;
5. collect the liquid under leaving, be used for the RT-PCR reaction.
Wherein, first optimal technical scheme of the present invention is that described method extracting post is to have one deck to play the film of physical barriers in the extracting column bottom.
Second optimal technical scheme of the present invention is that the concentration of the aqueous solution of described chelex-100 is 2～50g/100ml, more preferably 3～10g/100ml.
The 3rd optimal technical scheme of the present invention is that the concentration of the EDPC aqueous solution of described chelex-100 is 2～50g/100ml, preferred 3～10g/100ml.
The 4th optimal technical scheme of the present invention is that described Heating temperature is 90～100 ℃, preferred 90～95 ℃.
The 5th optimal technical scheme of the present invention is that be 5～30 minutes described heat-up time, preferred 8～10 minutes.
The 6th optimal technical scheme of the present invention is that the described centrifugal time is 2～120 seconds, preferred 10～30 seconds.
The 7th optimal technical scheme of the present invention does, described centrifugal rotation speed is 5000～20000 rev/mins, preferred 10000～20000 rev/mins, and more preferably 15000～20000 rev/mins.
Wherein, the biological specimen among the present invention is selected from pathological tissue paraffin section sample, blood cake seminal stain or semen sample, tissue juice sample, CSF sample, blood sample, serum sample, plasma sample, urine specimen, hydrops sample, sample of hair, histocyte sample; When biological specimen is histocyte sample or paraffin section, in step 1, adopt grinding rod that biological tissue is ground, when extracting RNA; Then need grind on ice; To reduce the RNA degraded, the release that improves RNA in the biological tissue improves checkability.
Technical superiority of the present invention is:
1. the inventive method is simple, and the time is short, and does not use harmful organic solvent.Generally extracting DNA needs are first from paraffin-embedded tissue in the traditional method remove YLENE with absolute ethyl alcohol then with the YLENE dewaxing, and the used time is long; Process is loaded down with trivial details; At least need 3～4 hours ability to accomplish, and YLENE is organic solvent, harmful.After having taken off wax, sample need pass through the processing of Proteinase K again, and to remove protein, this process also needs 3～4 hours, so DNA in the usual method extracting paraffin-embedded tissue gets into follow-up PCR reaction, needs more than 6～8 hours.Of the present invention method is simple, do not use volatility harmful organic solvent YLENE, can not damage the experimenter, and required time shortens dramatically, and minute can accomplish surplus only needing 10;
2. the present invention adopts heat to extract, and not only makes the paraffin liquefy, and makes the protein denaturation in the tissue, and DNA or RNA are released out, and having saved needs 3～4 hours process of Proteinase K hydrolysis needs;
3. liquid paraffin and chelex-100 can cause detrimentally affect to PCR or RT-PCR reaction usually; Through the present invention; Can liquid paraffin and chelex-100 be trapped on the film of extracting post; Thereby eliminated liquid paraffin and chelex-100 detrimentally affect, improved the sensitivity of PCR or RT-PCR reaction PCR or RT-PCR reaction;
4. still do not adopt at present Chelex-100 to extract the report of RNA, the contriver has proposed simply quick, the respond well method of from biological specimen, extracting RNA of a kind of Chelex-100 of employing in the present invention.
Description of drawings
Fig. 1 adopts the two kinds of extractive genomic dna agarose gel electrophoresis of method figure among the embodiment 1; Wherein, M represents molecular weight marker; 1 representative be with the extractive genomic dna agarose gel electrophoresis of test kit figure, 2 what represent is with the extractive genomic dna agarose gel electrophoresis of method of the present invention figure.
Following specific embodiments of the invention is done further explanation and explanation, but content of the present invention is not constituted restriction.The used reagent of the present invention is the commercial reagent, and used laboratory apparatus is the laboratory common instrument.
Embodiment
Embodiment 1
1. extract the DNA in the paraffin-embedded tissue section, 3 samples of every kind of method extracting, parallel comparison with two kinds of methods.
1) DNA during the paraffin organization section DNA extraction test kit extracting paraffin-embedded tissue of producing with Beijing hundred Tyke Bioisystech Co., Ltd is cut into slices
A. get medical paraffin (model: model: BX12M311398, the long-range Science and Technology Ltd. in Chinese and Western, Beijing produces) investing tissue's section of 2 5um and put into the centrifuge tube of 1.5mL.
B. add 1ml YLENE, concussion mixing, 55 ℃ of water-baths 10 minutes.13000 changeed 5 minutes, inhaled and abandoned supernatant.
C. repeating the 2nd goes on foot one to three time.
D. add the 1ml absolute ethyl alcohol, the concussion mixing.13000 changeed 5 minutes, inhaled and abandoned supernatant.
E. repeated for the 4th step once.
F. with the sample oven dry that settles down.
G. add 200 μ l Buffer 1, mixing.
H. add 25 μ l Proteinase K, the concussion mixing.55 ℃ of water-bath digestion are spent the night.
I.13000 left the heart 5 minutes, supernatant is transferred in the clean 1.5mL centrifuge tube.
J. add 220 μ l Buffer 2, the concussion mixing.70 ℃ of water-baths 10 minutes
K. add 220 μ l absolute ethyl alcohols, mixing.
L. the extracting post is put into collection tube, move into the mixing liquid in the 5th step, room temperature left standstill 3 minutes.
M.13000 leave the heart 2 minutes, and changed liquid over to pillar again, cross post again.
N.13000 leave the heart 2 minutes, and abandoned liquid.
O. move into pillar in the new collection tube, add 500 μ l Buffer 3.
P. move into pillar in the new collection tube, 13000 left the heart 1 minute, abandoned liquid.Add 600 μ l Wash Buffer.(Wash Buffer adds an amount of absolute ethyl alcohol in advance by packing)
Q.13000 leave the heart 1 minute, and abandoned liquid.Add 600 μ l DNA Wash Buffer.
R.13000 leave the heart 1 minute, and abandoned liquid.13000 left the heart 2 minutes, volatilization 4-5 minute of uncapping.
S., move into pillar in the centrifuge tube with cover of 1.5mL, in the adding 40 μ l Elution Buffer of film central authorities (70 ℃ of preheatings of palpus).Room temperature left standstill 3 minutes.
T.13000 left the heart 1 minute, and collected liquid, get 5ul and be used for the PCR reaction.
2) DNA in the method extracting paraffin-embedded tissue section of the present invention
A. get medical paraffin (model: model: BX12M311398, the long-range Science and Technology Ltd. in Chinese and Western, Beijing produces) investing tissue's section of 2 5um;
B. place the solution 1 of 200ul, described solution is the aqueous solution of 10%chelex-100 (sigma company);
C.90 heated 20 minutes under ℃ condition;
D. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 12000 rev/mins centrifugal 30 seconds; Described extracting post is:
E. collect the liquid under leaving, get 5ul and be used for the PCR reaction.
2.DN A authentication method:
2.1 the agarose gel electrophoresis of DNA quality is identified:
Because the dna content in the paraffin section is lower; Can not detect the amount of DNA with plain agar sugar gel electrophoresis method, so will use every kind of extractive three sample genomic dnas of method, respectively get 20 μ L and mix; After vacuum is taken out part moisture; Add appearance on the DNA loading buffer mixing of 5 μ L, 80V electrophoresis 30min in 2% sepharose, observation post extracts genomic quality of DNA and mug under ultraviolet gel imaging appearance.Fig. 1 is the two kinds of extractive genomic dna agarose gel electrophoresis of method figure; Wherein, M represents molecular weight marker, 1 representative be with the extractive genomic dna agarose gel electrophoresis of test kit figure, 2 what represent is with the extractive genomic dna agarose gel electrophoresis of method of the present invention figure.
2.2 the genomic concentration of DNA that fluorescence quantitative PCR detection is extracted
Select primer and TaqMan fluorescent probe, adopt Primer Express software to design voluntarily to people β-2actin genomic dna, synthetic by Shanghai bio-engineering corporation (Sangong).Primer sequence is seen table 1.
Table 1: the sequence table of primer and probe
| Upstream primer | 5′-GGACCTGACTGACTACCTCATGAA-3′ |
| Downstream primer | 5′-CTTAATGTCACGCACGATTTCC-3′ |
| Probe | 5′Fam-CACCGAGCGCGGCTACAGCTTC-TAMARA?3′ |
PCR reaction system: template DNA 2 μ l, 10 * buffer (no Mg2+) 2.5ul, 2.5mmoL/L dNTP 2ul; 25mmoL/L MgCl2 1.5ul, primer 10mmol/L 0.5ul, 10mmol/L probe 0.4ul; 5U/ul Taq polysaccharase 0.5 μ l, remainder is supplied by the sterilization deionized water
The pcr amplification condition: 94 ℃ of 3min, 94 ℃ of 15s, 60 ℃ of 60s, 40 circulations, 60 ℃ are detected fluorescence, and the positive, negative control are all established in each PCR reaction.
Selected three samples altogether, each sample repeats 5 times, uses the amount of β in each sample of fluorescence quantitative PCR detection people-2actin genomic dna respectively, uses the Ct value representation, compares the quality and quantity of two kinds of DNA that method for extracting obtained, and experimental result is as shown in table 2.
Visible from table 2, the extractive Ct value of Ct value comparison reagent kit of the β-2actin that genomic dna increased that obtains with the inventive method extracting is little, and all comparison reagent kit is extractive good to point out the genomic dna content that obtains with the inventive method extracting and quality.
Table 2: adopt two kinds of methods to extract the Ct value of DNA
Embodiment 2
1. the seminal stain sample is placed the aqueous solution of chelex-100, the concentration of the aqueous solution of described chelex-100 is 20g/100ml;
2.95 heating is 10 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 20000 rev/mins, centrifugal 20 seconds;
4. collect the liquid under leaving, be used for the PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 1 adopted in PCR reaction, and obtained similar experimental result.
Embodiment 3
1. the tissue juice sample is placed the aqueous solution of chelex-100, the concentration of the aqueous solution of described chelex-100 is 10g/100ml;
2.100 heating is 50 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 15000 rev/mins, centrifugal 30 seconds;
4. collect the liquid under leaving, be used for the PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 1 adopted in PCR reaction, and obtained similar experimental result.
Embodiment 4
1. after the histocyte sample being ground with grinding rod, place the aqueous solution of chelex-100, the concentration of the aqueous solution of described chelex-100 is 15g/100ml;
2.80 heating is 10 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 15000 rev/mins, centrifugal 30 seconds;
4. collect the liquid under leaving, be used for the PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 1 adopted in PCR reaction, and obtained similar experimental result.
Embodiment 5
1. the blood cake sample is placed the aqueous solution of chelex-100, the concentration of the aqueous solution of described chelex-100 is 20g/100ml;
2.95 heating is 10 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 20000 rev/mins, centrifugal 20 seconds;
4. collect the liquid under leaving, be used for the PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 1 adopted in PCR reaction, and obtained similar experimental result.
Embodiment 6
1. the RNA during the rapid extraction paraffin-embedded tissue is cut into slices:
Medical paraffin (model: BX12M311398, the long-range Science and Technology Ltd. in Chinese and Western, Beijing produces) investing tissue's section of a) getting 3 5um is put into the centrifuge tube of 1.5mL on ice after grinding;
B) place the solution 1 of 200ul, described solution is the DEPC aqueous solution of 10%chelex-100;
C) heated 20 minutes under 90 ℃ of conditions;
D) the extracting column jecket is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting column jecket, 12000 rev/mins centrifugal 30 seconds;
E) liquid under collection is left is got 5ul and is used for the RT-PCR reaction.
2. fluorescence quantitative RT-RCR detects the concentration of the RNA that extracts
Select to people GAPDH primer and TaqMan fluorescent probe, adopt Primer Express software to design voluntarily, synthetic by Shanghai bio-engineering corporation (Sangong).Primer sequence is seen table 1.
Table 1 is the nucleotide sequence of primer, probe:
| Upstream primer | 5′-CATCTTCCAGGAGCGAGA-3′ |
| Downstream primer | 5′-TGTTGTCATACTTCTCAT-3′ |
| Probe | 5′Fam-CCTCACCACCATGGAGAAGGCT-TAMARA?3′ |
Contain in the 40 μ L reaction systems: 2.5 μ L, 10 * PCR reaction buffer (Mg2+free), 3mmol/L Mg2+, 0.125mmol/L dNTP; 0.25 μ mol/L primer, 0.156 μ mol/L probe, polysaccharase 2U; UNG enzyme 0.2U; RT enzyme 100u, the template amount is 3 μ L, make up water to 40 μ L.
Pcr amplification condition: 42 ℃ of 30min, 94 ℃, 5min; 94 ℃ of 15s, 60 ℃ of 60s, 40 circulations, 60 ℃ are detected fluorescence, and the positive, negative control are all established in each PCR reaction.
The mRNA amplification of RT-PCR to GAPDH carried out in 8 paraffin-embedded tissue sections of picked at random, and each sample can both amplify GAPDH, and it is feasible fast pointing out this method, sees table 2.
Table 2 is the Ct value of sample of the present invention:
| Mark this shop | The Ct value |
| 1 | 32.1382 |
| 2 | 32.694 |
| 3 | 29.1025 |
| 4 | 28.9604 |
| 5 | 24.662 |
| 6 | 24.5847 |
| 7 | 21.0877 |
| 8 | 21.1414 |
Embodiment 7
1. the seminal stain sample is placed the DEPC aqueous solution of 100 μ l chelex-100, the concentration of the DEPC aqueous solution of described chelex-100 is 20g/100ml;
2.95 heating is 10 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 20000 rev/mins, centrifugal 20 seconds;
4. collect the liquid under leaving, be used for the RT-PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 6 adopted in RT-PCR reaction, and obtained similar experimental result.
Embodiment 8
1. the tissue juice sample is placed the DEPC aqueous solution of 200 μ l chelex-100, the concentration of the DEPC aqueous solution of described chelex-100 is 10g/100ml;
2.100 heating is 50 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 15000 rev/mins, centrifugal 30 seconds;
4. collect the liquid under leaving, be used for the RT-PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 6 adopted in RT-PCR reaction, and obtained similar experimental result.
Embodiment 9
1. after the histocyte sample being ground with grinding rod on ice, place the DEPC aqueous solution of 400 μ l chelex-100, the concentration of the DEPC aqueous solution of described chelex-100 is 15g/100ml;
2.80 heating is 10 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 15000 rev/mins, centrifugal 30 seconds;
4. collect the liquid under leaving, be used for the RT-PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 6 adopted in RT-PCR reaction, and obtained similar experimental result.
Embodiment 10
1. the blood cake sample is placed the DEPC aqueous solution of 800 μ l chelex-100, the concentration of the DEPC aqueous solution of described chelex-100 is 20g/100ml;
2.95 heating is 10 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 20000 rev/mins, centrifugal 20 seconds;
4. collect the liquid under leaving, be used for the RT-PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 6 adopted in RT-PCR reaction, and obtained similar experimental result.
Claims (4)
1. the method for a rapid extraction DNA from biological specimen is characterized in that, described method may further comprise the steps:
(1) biological specimen that takes a morsel, said biological specimen is selected from pathological tissue paraffin section sample, blood cake seminal stain or semen sample, tissue juice sample, CSF sample, blood sample, serum sample, plasma sample, urine specimen, hydrops sample, sample of hair, histocyte sample;
(2) biological specimen is placed the aqueous solution of chelex-100, the concentration of the aqueous solution of described chelex-100 is 1～80g/100ml;
Heated 1～50 minute under (3) 80～100 ℃ of conditions;
(4) the extracting post is put into centrifuge tube, the liquid that obtains in the step (3) is joined in the extracting post, 1000～20000 rev/mins, centrifugal 2～120 seconds;
(5) liquid under collection is left is used for the PCR reaction.
2. the method for a rapid extraction RNA from biological specimen is characterized in that, described method may further comprise the steps:
(1) biological specimen that takes a morsel, said biological specimen is selected from pathological tissue paraffin section sample, blood cake seminal stain or semen sample, tissue juice sample, CSF sample, blood sample, serum sample, plasma sample, urine specimen, hydrops sample, sample of hair, histocyte sample;
(2) place the DEPC aqueous solution of 1～80%chelex-100;
Heated 1～50 minute under (3) 80～100 ℃ of conditions;
(4) the extracting column jecket is put into centrifuge tube, the liquid that obtains in the step (3) is joined in the extracting column jecket, 1000～20000 rev/mins, centrifugal 2～120 seconds;
(5) liquid under collection is left is used for the RT-PCR reaction.
3. method according to claim 1 and 2 is characterized in that, described extracting post is to have one deck to play the film of physical barriers in the extracting column bottom.
4. method according to claim 1 and 2 is characterized in that, the concentration of the aqueous solution of described chelex-100 is 2～50g/100ml, preferred 3～10g/100ml; The concentration of the DEPC aqueous solution of described chelex-100 is 2～50g/100ml, preferred 3～10g/100ml.
A) method according to claim 1 and 2 is characterized in that, described Heating temperature is 90～100 ℃, preferred 90～95 ℃.
B) method according to claim 1 and 2 is characterized in that, be 5～30 minutes described heat-up time, preferred 8～10 minutes.
C) method according to claim 1 and 2 is characterized in that, the described centrifugal time is 2～120 seconds, preferred 10～30 seconds.
D) method according to claim 1 and 2 is characterized in that, described centrifugal rotation speed is 5000～20000 rev/mins, preferred 10000～20000 rev/mins, and more preferably 15000～20000 rev/mins.
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| CN103757095A (en) * | 2013-10-19 | 2014-04-30 | 中国医科大学 | Method for distinguishing individual in mixed seminal stain by single sperm capture and mitochondrial DNA typing |
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