CN108795928A - A method of for DNA and RNA in separation and Extraction cell - Google Patents
A method of for DNA and RNA in separation and Extraction cell Download PDFInfo
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- CN108795928A CN108795928A CN201810709793.2A CN201810709793A CN108795928A CN 108795928 A CN108795928 A CN 108795928A CN 201810709793 A CN201810709793 A CN 201810709793A CN 108795928 A CN108795928 A CN 108795928A
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- dna
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- 238000000034 method Methods 0.000 title claims abstract description 57
- 238000000605 extraction Methods 0.000 title claims abstract description 22
- 238000000926 separation method Methods 0.000 title claims abstract description 13
- 238000001556 precipitation Methods 0.000 claims abstract description 69
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 55
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000012071 phase Substances 0.000 claims abstract description 16
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 15
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 15
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 15
- 238000000197 pyrolysis Methods 0.000 claims abstract description 12
- 239000002904 solvent Substances 0.000 claims abstract description 6
- 239000012074 organic phase Substances 0.000 claims abstract description 5
- 230000002101 lytic effect Effects 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 65
- 238000005119 centrifugation Methods 0.000 claims description 38
- 210000004027 cell Anatomy 0.000 claims description 30
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 29
- 235000019441 ethanol Nutrition 0.000 claims description 28
- 239000002244 precipitate Substances 0.000 claims description 27
- 238000002156 mixing Methods 0.000 claims description 26
- 239000012459 cleaning agent Substances 0.000 claims description 22
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 17
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 15
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 14
- 239000000047 product Substances 0.000 claims description 11
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 9
- 238000005336 cracking Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 235000011091 sodium acetates Nutrition 0.000 claims description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical group ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- 229960004063 propylene glycol Drugs 0.000 claims description 7
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 5
- 239000003161 ribonuclease inhibitor Substances 0.000 claims description 5
- 239000001632 sodium acetate Substances 0.000 claims description 5
- 235000017281 sodium acetate Nutrition 0.000 claims description 5
- 229920001612 Hydroxyethyl starch Polymers 0.000 claims description 4
- 241000288906 Primates Species 0.000 claims description 4
- 238000000432 density-gradient centrifugation Methods 0.000 claims description 4
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims description 4
- 229940050526 hydroxyethylstarch Drugs 0.000 claims description 4
- 239000004615 ingredient Substances 0.000 claims description 4
- 210000004962 mammalian cell Anatomy 0.000 claims description 4
- MFESCIUQSIBMSM-UHFFFAOYSA-N I-BCP Chemical class ClCCCBr MFESCIUQSIBMSM-UHFFFAOYSA-N 0.000 claims description 3
- 239000005725 8-Hydroxyquinoline Substances 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- NBQNWMBBSKPBAY-UHFFFAOYSA-N iodixanol Chemical compound IC=1C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C(I)C=1N(C(=O)C)CC(O)CN(C(C)=O)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NBQNWMBBSKPBAY-UHFFFAOYSA-N 0.000 claims description 2
- 229960004359 iodixanol Drugs 0.000 claims description 2
- 229960003540 oxyquinoline Drugs 0.000 claims description 2
- MCJGNVYPOGVAJF-UHFFFAOYSA-N quinolin-8-ol Chemical compound C1=CN=C2C(O)=CC=CC2=C1 MCJGNVYPOGVAJF-UHFFFAOYSA-N 0.000 claims description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 2
- 239000002342 ribonucleoside Substances 0.000 claims 1
- 239000000284 extract Substances 0.000 abstract description 4
- 108020004414 DNA Proteins 0.000 description 96
- 239000000243 solution Substances 0.000 description 32
- 239000000523 sample Substances 0.000 description 20
- 239000006228 supernatant Substances 0.000 description 14
- 239000008346 aqueous phase Substances 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 11
- 230000000694 effects Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000007710 freezing Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 239000007984 Tris EDTA buffer Substances 0.000 description 4
- 238000010936 aqueous wash Methods 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 230000001376 precipitating effect Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 125000003647 acryloyl group Chemical group O=C([*])C([H])=C([H])[H] 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 210000004295 hippocampal neuron Anatomy 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- -1 riboside compound Chemical class 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 229910001415 sodium ion Inorganic materials 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 235000011083 sodium citrates Nutrition 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical class [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 125000005287 vanadyl group Chemical group 0.000 description 1
- 230000010148 water-pollination Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to biotechnologies, in particular to a kind of method for DNA and RNA in separation and Extraction cell.Described includes a) obtaining pyrolysis product using Trizol lytic cell samples release nucleic acid；B) it is centrifuged after contacting the pyrolysis product at least one organic extraction solvent, so that the pyrolysis product is layered to obtain upper layer RNA water phases, the batt layer of middle level DNA and lower layer's organic phase；C) batt layer of the RNA water phases and the DNA precipitated respectively, cleaned；Wherein, when carrying out the precipitation, DNA precipitation reagents used include 1,2-PD.The problem of DNA yields are low when this method can effectively avoid while extract DNA and RNA, purity difference.
Description
Technical field
The present invention relates to biotechnologies, in particular to a kind of for DNA and RNA in separation and Extraction cell
Method.
Background technology
It is related to complicated multi-step from the art methods of the concentration of cell origin material, extracting and developing and purification of nucleic acid
Process.When handling some biological samples, or carrying out clinical examination, the sample size obtained is usually very rare, thus is
Make full use of sample, it usually needs while extracting DNA and RNA.
In the prior art, the method for extracting DNA and RNA simultaneously from sample is generally Trizol cracking process.Trizol is tried
Agent is a kind of RNA extracts reagents of maturation suitable for the quick separating RNA from cell and tissue.Trizol reagents have multicomponent
Centrifugation, it is maximum special compared with other methods such as guanidine thiocyanate/phenol method, phenol/SDS methods, guanidine hydrochloride method, guanidine thiocyanate method
Point be can detach simultaneously a sample RNA DNA protein.Trizol makes sample homogenization, and cell cracking, dissolving is into the cell
Inclusion, while the integrality because RNA can be kept containing RNase inhibitor.After chloroform centrifugation is added, solution is divided into water phase and has
Machine phase, RNA is in water phase.Take out the recyclable RNA of water phase isopropanol precipitating；With the recyclable DNA of ethanol precipitation middle layer；With different
Propyl alcohol precipitates the recyclable protein of organic phase.
However, effect is but often not satisfactory when Trizol extracts DNA, DNA concentration and purity have larger promotion empty
Between.
In view of this, special propose the present invention.
Invention content
The purpose of the present invention is to provide a kind of method for DNA and RNA in separation and Extraction cell, this method is extracted
The nucleic acid purity that arrives is more preferable, yield higher, and has better extraction efficiency.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
The present invention relates to a kind of methods for DNA and RNA in separation and Extraction cell, including：
A) pyrolysis product is obtained using Trizol lytic cell samples release nucleic acid；
B) it is centrifuged after contacting the pyrolysis product at least one organic extraction solvent, so that the pyrolysis product point
Layer obtains upper layer RNA water phases, the batt layer of middle level DNA and lower layer's organic phase；
C) batt layer of the RNA water phases and the DNA precipitated respectively, cleaned；
Wherein, when carrying out the precipitation, DNA precipitation reagents used include 1,2-PD.
Specific implementation mode
The present invention can by subsequently some embodiments of the invention is described and wherein included embodiment it is detailed
Thin content and be easier to be appreciated that.
Before being further discussed below the present invention, it should be apparent that the present invention is not limited in the particular embodiment, because
It is necessarily various for these embodiments.Also it should be apparent that term as used in this specification is merely to illustrate particular implementation
Scheme, rather than as limitation, because the scope of the present invention will be defined only in the appended claims.
The present invention relates to a kind of methods for DNA and RNA in separation and Extraction cell, including：
A) pyrolysis product is obtained using Trizol lytic cell samples release nucleic acid；
B) it is centrifuged after contacting the pyrolysis product at least one organic extraction solvent, so that the pyrolysis product point
Layer obtains upper layer RNA water phases, the batt layer of middle level DNA and lower layer's organic phase；
C) batt layer of the RNA water phases and the DNA precipitated respectively, cleaned；
Wherein, when carrying out the precipitation, DNA precipitation reagents used include 1,2-PD.
Preferably, method as described above also includes the second of ethyl alcohol and/or pH=5~5.4 in the DNA precipitation reagents
Sour sodium.
Preferably, method as described above, the DNA precipitation reagents are mixed to get by the ingredient of following volumes part：
1~3 part of 1,2- propylene glycol, 6~10 parts of ethyl alcohol, pH=5~5.4 1~1.5 part of 1.5M sodium acetates；
In some embodiments, the DNA precipitation reagents are mixed to get by the ingredient of following volumes part：
1.5~2.5 parts of 1,2- propylene glycol, 7~9 parts of ethyl alcohol, pH=5~5.4 1.1~1.4 parts of 1.5M sodium acetates；
In some embodiments, the DNA precipitation reagents are mixed to get by the ingredient of following volumes part：
2 parts of 1,2- propylene glycol, 8 parts of ethyl alcohol, pH=5~5.4 1.2 parts of 1.5M sodium acetates.
The buffer reagent of the 1.5M sodium acetates of pH=5~5.4 actually sodium acetate-acetic acid.
RNA or DNA is polar molecule, and nucleic acid contains a large amount of phosphate group (PO3-), it can be with polar hydrone
Electrostatic interaction, thus it makes it easier to be dissolved in the water.
The precipitation of DNA mostly uses absolute ethyl alcohol or isopropanol in the prior art.Ethyl alcohol and isopropanol all have hydroxyl, can be with
Nucleic acid molecules fight for water, this is the reason that it promotes nucleic acid molecules precipitation.The advantages of ethyl alcohol is, and second few to precipitation of salts
The easy evaporative removal of alcohol, does not influence later experiment.But it is some larger that the polarity of ethyl alcohol compares isopropanol, required body when for precipitating
Product is more.Isopropanol polarity is small, and required volume is small and speed is fast, low suitable for concentration, and the precipitation of the big DNA sample of volume, but
Isopropanol easily makes salt (such as NaCl, sucrose) be co-precipitated with DNA.
For disadvantages mentioned above, 1,2-PD is added in the present application selection in precipitation system.
1,2-PD has lower polarity, and it possesses more hydroxyls, is more favorable for DNA and more precipitates, this
Invention is mixed using 1,2-PD with the ethyl alcohol of appropriate amount, only to precipitate salt under the premise of ensureing Precipitation Potential more.
Extraction environment slant acidity can be adjusted using sodium acetate, to effective neutralization precipitation environment, control precipitation system is suitable
It is carried out under suitable dielectric constant.In addition, the sodium ion of suitable concentration can neutralize the charge neutralized on sugared phosphate backbones, make molecule
Hydrophily reduces, therefore solubility in water is lower.
When being extracted due to Trizol, the DNA content of middle layer is minimum, and it is usually very low to often lead to DNA ultimate yields.It uses
The precipitation reagent that the present invention improves can greatly improve DNA yields.
Preferably, method as described above, when being precipitated to the batt layer of the DNA with the DNA precipitation reagents,
It first mixes well, 15min~30min is stood at 18 DEG C~30 DEG C, it is also an option that 17min, 20min, 23min, 25min
Or 28min.
Preferably, method as described above, the volume ratio of the batt layer of the DNA precipitation reagents and the DNA is 0.6~
0.8:0.8~1.2；It is also an option that 0.7：1.
The present invention increases sedimentation effect using 1,2-PD and sodium acetate, thus lesser amount of DNA precipitations examination can be used
Agent, to reduce the introducing of propylene glycol and sodium ion.
Preferably, method as described above, when being cleaned to the DNA after precipitation, it is clear that cleaning agent used is divided into first
Lotion and the second cleaning agent；
First cleaning agent is absolute ethyl alcohol；
Second cleaning agent is the ethanol water of 70v/v%~80v/v%, it is also an option that 72v/v%, 74v/
The ethanol water of v%, 75v/v%, 76v/v% or 78v/v%.
Preferably, method as described above, with first cleaning agent or second cleaning agent to the DNA after precipitation into
When row cleaning, directly centrifuged after precipitating mixing with upper step.
Preferably, method as described above, it is described when being cleaned to the DNA after precipitation with first cleaning agent
Centrifugation carries out at 18 DEG C~30 DEG C；
The condition of centrifugation is 9000rpm~11000rpm, 10min~15min.
The condition of centrifugation can also select 10000rpm, 12min.
Preferably, method as described above, when being cleaned to the DNA after precipitation with second cleaning agent, centrifugation
Condition be 11000rpm~13000rpm, 3min~7min；
The condition of centrifugation can also select 12000rpm, 5min.
1,2-PD is not volatile compared to for isopropanol, thus the method that the present invention uses the elution of two steps, exhausted big portion
Divide 1,2- propylene glycol that can all be eluted in the process.
Centrifugation carries out at 18 DEG C~30 DEG C when eluting for the first time, can further promote 1,2-PD and remaining phenol
It is mixed into absolute ethyl alcohol and is removed.
Preferably, method as described above, the RNA precipitate reagent includes isopropanol.
Preferably, method as described above, the RNA precipitate reagent further include that RNase inhibitor is appropriate.
Preferably, method as described above, the RNase inhibitor are selected from 8-hydroxyquinoline, guanidinium isothiocyanate, vanadyl core
It is one kind of multiple in riboside compound, RNasin, SDS, beta -mercaptoethanol.
Preferably, method as described above, when being precipitated to the RNA water phases with the RNA precipitate reagent, fully
10min~20min is stood after mixing at 18 DEG C~30 DEG C, it is also an option that 12min, 14min, 16min or 18min.
Preferably, the volume ratio of method as described above, the RNA precipitate reagent and the RNA water phases is 0.8~1.2:
0.8~1.2；It is also an option that 1：1.
Preferably, method as described above, when being cleaned to the RNA after precipitation, cleaning agent used is 70v/v%
The ethanol water of~80v/v%；
In some embodiments, cleaning agent used is 72v/v%, 74v/v%, 75v/v%, 76v/v% or 78v/v%
Ethanol water.
Preferably, method as described above, the cleaning are 1~2 time.
Preferably, method as described above when being cleaned to the RNA after precipitation with the cleaning agent, is precipitated with upper step
2 DEG C~6 DEG C incubation 1min~5min, centrifugation after mixing；
The condition of centrifugation is 2 DEG C~6 DEG C, 11000rpm~13000rpm, 3min~7min；
The condition of centrifugation is also an option that 4 DEG C, 12000rpm, 5min.
Preferably, method as described above also contains linear when cracking PBMC samples using Trizol, in cracking system
Change acrylamide.
Acrylamide solution is linearized, is a kind of coprecipitator, contributes to the precipitation of nucleic acid purification process amplifying nucleic acid.
Preferably, the condition of method as described above, the cracking is：
Trizol reagents and the cell sample mixing are first used, linearisation acryloyl is added after standing 15min~25min
Amine stands 3min~8min after mixing；
The condition of the cracking is also an option that：
Trizol reagents and the cell sample mixing are first used, linearisation acryloyl is added after standing 18min~22min
Amine stands 4min~6min after mixing；
Or, first using Trizol reagents and the cell sample mixing, linearisation acrylamide is added after standing 20min,
5min is stood after mixing.
Preferably, method as described above, the organic extraction solvent are selected from chloroform or the bromo- 3- chloropropanes of 1-；More preferably
For the bromo- 3- chloropropanes (BCP) of 1-.
BCP low toxicities, boiling point is high, not volatile (BCP, bp:142-145℃；chloroform,bp:60.5-61.5 DEG C),
Using upper safer, it need not move in draught cupboard and operate.
Preferably, method as described above, the centrifugation are 10000rpm~14000rpm under conditions of 2 DEG C~6 DEG C
Centrifuge 10min~20min；
The centrifugation can also be under conditions of 2 DEG C~6 DEG C, and 11000rpm~13000rpm centrifuges 13min~17min；
The centrifugation can also be under conditions of 4 DEG C, and 12000rpm centrifuges 15min.
Preferably, method as described above, the cell sample are zooblast sample.
Preferably, method as described above, the zooblast sample are mammalian cell sample.
Preferably, method as described above, the mammalian cell sample are primates zooblast sample.
Preferably, method as described above, the primates zooblast sample are the cell sample of people.
This method is particularly suitable for treasuring the separation of cell sample amplifying nucleic acid, such as PBMC samples.
The separation of PBMC can be used that well known to a person skilled in the art method progress.Preferably, method as described above, institute
It states PBMC samples and passes through Ficoll-Hypaque density-gradient centrifugation methods, Percoll density-gradient centrifugation methods, Iodixanol ladders
It is isolated to spend any one of centrifugal process, hydroxyethyl starch (HES) centrifugal method.
PBMC separation methods are illustrated
(a) whole blood of heparin lithium anti-freezing is numbered, trim, 2000rpm/min centrifuges 10min.Blood plasma is taken out, in -80
It DEG C freezes.Remaining haemocyte adds PBS to l0ml, mixing.
(b) 2 centrifuge tubes are taken, often pipe plus 4m1 lymphocyte separation mediums, it is thin to be added slowly to lymph by above-mentioned mixed whole blood
The upper layer of born of the same parents' separating liquid makes the two form a clearly interface.
(c) trim, 2500rpm/min centrifugation 20min after obtain 4 cellular layers, be followed successively by from top to bottom diluted blood plasma,
PBMC, granulocyte, red blood cell.
(d) it takes in second layer tunica albuginea layer to centrifuge tube, adds PBS to l0ml, trim, 1500rpm/min centrifuges 10min.
(e) supernatant is abandoned, adds PBS to 10ml to be uniformly mixed, and carry out cell count.
If isolated PBMC need not be used at once, further include：
(f) 1500rpm/min centrifuges 5min, abandons supernatant, and gently bullet dissipates cell mass, by 1:1 plus A, B liquid, mixing, with 5 ×
106~1 × 107/ pipe is positioned in freezing storing box rapidly.
(g) freezing storing box is put into -80 DEG C of refrigerators.
(h) next day moves into freeze-stored cell in liquid nitrogen.
Points for attention：
(a) pay attention to whole process sterile working.
(b) pay attention to trim when centrifuging, prevent shakiness from causing cell centrifugal effect bad.
(c) whole blood is noticed that speed is slow toward the lymphocyte separation medium upper layer added-time, forms a clearly liquid levels layer,
It prevents from mixing, influences separating effect.
(d) A, B liquid are mixed into frozen stock solution, for freezing for cell.
(e) A liquid is cow's serum；B liquid is the mixing of cow's serum and DMSO, ratio 4：1.
(f) cell not put the long time in freezing storing box, influenced the survival service life of cell, be transplanted in liquid nitrogen container as early as possible
It preserves.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Embodiment 1
A kind of method of extraction Hippocampal Neuron Cells nucleic acid is present embodiments provided, including：
1. taking out Hippocampal Neuron Cells sample, 1mL Trizol reagents are added, under piping and druming 12~16,18 DEG C of standings
30min is added 1 μ L and linearizes acrylamide solution, overturns mixing, 18 DEG C of standing 8min；
2. the BCP reagents of 100 μ L precoolings are added, under firmly rocking 20~30 with hand, wait for that solution is creamy white state, 18 DEG C
Stand 18min；
3. 4 DEG C of centrifugations of 10000rpm 20min prepare the EP pipes of corresponding labeled RNA printed words；
4. after centrifugation, solution is divided into three layers in EP pipes, and upper layer is the aqueous phase solution containing RNA, and centre is to contain genome
The batt layer of DNA, lower layer are red organic solution layer.
It is careful to take out about 500 μ L upper strata aqueous phase solution, move to the EP pipes of new labeled RNA printed words；
It is careful to take out about 300 μ L middle layer aqueous phase solutions, move to the EP pipes of new marker DNA printed words.
5. being separately added into 1 μ L RNasin and 500 μ L isopropanols in RNA EP pipes, mixing, 18 DEG C of standing 20min are overturned.
4 DEG C of 12000rpm 15min are centrifuged, at this time visible EP bottom of the tube white precipitate.It is careful that supernatant is sucked out, it discards.
RNA precipitate is washed 2 times with 1mL 70v/v% ethyl alcohol, i.e., is first incubated 3min, 4 DEG C of centrifugations of rear 12000rpm 5min.
After RNA precipitate washes, lies low to be positioned on toilet paper and dry (40~60min) afterwards with 30 μ L ddH2O dissolves, and 55
DEG C water-bath 30min, nano drop survey RNA concentration and purity.
6. 210 μ L of DNA precipitation reagents are added in DNA EP pipes, the DNA precipitation reagents are by 1~3 part of 1,2-PD, second
1~1.5 part of preparation of 1.5M sodium acetates of 6~10 parts of alcohol, pH=5~5.4 obtains.
It mixes well, cotton-shaped DNA precipitations visible at this time occur, 18 DEG C of standing 30min；
4 DEG C of 10000rpm 20min are centrifuged, at this time visible EP bottom of the tube white precipitate.It is careful that supernatant is sucked out, it discards.
DNA precipitations are washed once with absolute ethyl alcohol, and at 18 DEG C, 11000rpm centrifuges 10min；
After discarding supernatant, DNA precipitations continue to use the ethanol aqueous wash of 70v/v% primary, and the condition of centrifugation is
11000rpm, 7min, 4 DEG C or room temperature (18 DEG C~30 DEG C).
The EP pipes of DNA, which lie low to be positioned on toilet paper, dries (40~60min) afterwards with 30 μ L TE buffer dissolvings, 55 DEG C
Water-bath 30min, nano drop survey RNA concentration and purity.
Embodiment 2
A kind of method of extraction fibroblast nucleic acid is present embodiments provided, including：
1. taking out fibroblast sample, 1mL Trizol reagents are added, under piping and druming 12~16,30 DEG C of standing 15min add
Enter 1 μ L linearisation acrylamide solutions, overturns mixing, 30 DEG C of standing 3min；
2. the BCP reagents of 100 μ L precoolings are added, under firmly rocking 20~30 with hand, wait for that solution is creamy white state, 30 DEG C
Stand 12min；
3. 4 DEG C of centrifugations of 14000rpm 10min prepare the EP pipes of corresponding labeled RNA printed words；
4. after centrifugation, solution is divided into three layers in EP pipes, and upper layer is the aqueous phase solution containing RNA, and centre is to contain genome
The batt layer of DNA, lower layer are red organic solution layer.
It is careful to take out about 500 μ L upper strata aqueous phase solution, move to the EP pipes of new labeled RNA printed words；
It is careful to take out about 300 μ L middle layer aqueous phase solutions, move to the EP pipes of new marker DNA printed words.
5. being separately added into 1 μ L RNasin and 500 μ L isopropanols in RNA EP pipes, mixing, 30 DEG C of standing 12min are overturned.
4 DEG C of 12000rpm 15min are centrifuged, at this time visible EP bottom of the tube white precipitate.It is careful that supernatant is sucked out, it discards.
RNA precipitate is washed 2 times with 1mL 80v/v% ethyl alcohol, i.e., is first incubated 3min, 4 DEG C of centrifugations of rear 12000rpm5min.
After RNA precipitate washes, lies low to be positioned on toilet paper and dry (40~60min) afterwards with 30 μ L ddH2O dissolves, and 55
DEG C water-bath 30min, nano drop survey RNA concentration and purity.
6. 210 μ L of DNA precipitation reagents are added in DNA EP pipes, the DNA precipitation reagents are by 1~3 part of 1,2-PD, second
1~1.5 part of preparation of 1.5M sodium acetates of 6~10 parts of alcohol, pH=5~5.4 obtains.
It mixes well, cotton-shaped DNA precipitations visible at this time occur, 30 DEG C of standing 15min；
4 DEG C of 14000rpm 10min are centrifuged, at this time visible EP bottom of the tube white precipitate.It is careful that supernatant is sucked out, it discards.
DNA precipitations are washed once with absolute ethyl alcohol, and at 30 DEG C, 9000rpm centrifuges 15min；
After discarding supernatant, DNA precipitations continue to use the ethanol aqueous wash of 80v/v% primary, and the condition of centrifugation is
13000rpm, 3min, 4 DEG C or room temperature (18 DEG C~30 DEG C).
The EP pipes of DNA, which lie low to be positioned on toilet paper, dries (40~60min) afterwards with 30 μ L TE buffer dissolvings, 55 DEG C
Water-bath 30min, nano drop survey RNA concentration and purity.
Embodiment 3
A kind of method of extraction PBMC nucleic acid is present embodiments provided, including：
1. taking out PBMC samples, 1mL Trizol reagents are added, under piping and druming 12~16,1 μ L are added in 20 DEG C of standing 25min
Acrylamide solution is linearized, mixing, 20 DEG C of standing 4min are overturned；
2. the BCP reagents of 100 μ L precoolings are added, under firmly rocking 20~30 with hand, wait for that solution is creamy white state, 20 DEG C
Stand 4min；
3. 4 DEG C of centrifugations of 13000rpm 12min prepare the EP pipes of corresponding labeled RNA printed words；
4. after centrifugation, solution is divided into three layers in EP pipes, and upper layer is the aqueous phase solution containing RNA, and centre is to contain genome
The batt layer of DNA, lower layer are red organic solution layer.
It is careful to take out about 500 μ L upper strata aqueous phase solution, move to the EP pipes of new labeled RNA printed words；
It is careful to take out about 300 μ L middle layer aqueous phase solutions, move to the EP pipes of new marker DNA printed words.
5. being separately added into 1 μ L RNasin and 500 μ L isopropanols in RNA EP pipes, mixing, 20 DEG C of standing 15min are overturned.
4 DEG C of 12000rpm 15min are centrifuged, at this time visible EP bottom of the tube white precipitate.It is careful that supernatant is sucked out, it discards.
RNA precipitate is washed 2 times with 1mL 73v/v% ethyl alcohol, i.e., is first incubated 3min, 4 DEG C of centrifugations of rear 12000rpm5min.
After RNA precipitate washes, lies low to be positioned on toilet paper and dry (40~60min) afterwards with 30 μ L ddH2O dissolves, and 55
DEG C water-bath 30min, nano drop survey RNA concentration and purity.
6. 210 μ L of DNA precipitation reagents are added in DNA EP pipes, the DNA precipitation reagents are by 2.5 parts of 1,2-PD, second
1 part of preparation of 1.5M sodium acetates of 9 parts of alcohol, pH=5~5.4 obtains.
It mixes well, cotton-shaped DNA precipitations visible at this time occur, 20 DEG C of standing 12min；
4 DEG C of 12000rpm 12min are centrifuged, at this time visible EP bottom of the tube white precipitate.It is careful that supernatant is sucked out, it discards.
DNA precipitations are washed once with absolute ethyl alcohol, and at 18 DEG C~30 DEG C, 9000rpm centrifuges 15min；
After discarding supernatant, DNA precipitations continue to use the ethanol aqueous wash of 77v/v% primary, and the condition of centrifugation is
12000rpm, 6min, 4 DEG C or room temperature (20 DEG C).
The EP pipes of DNA, which lie low to be positioned on toilet paper, dries (40~60min) afterwards with 30 μ L TE buffer dissolvings, 55 DEG C
Water-bath 30min, nano drop survey RNA concentration and purity.
Embodiment 4
A kind of method of extraction PBMC nucleic acid is present embodiments provided, including：
1. taking out PBMC samples, 1mL Trizol reagents are added, under piping and druming 12~16,1 μ L are added in 25 DEG C of standing 25min
Acrylamide solution is linearized, mixing, 25 DEG C of standing 5min are overturned；
2. the BCP reagents of 100 μ L precoolings are added, under firmly rocking 20~30 with hand, wait for that solution is creamy white state, 25 DEG C
Stand 15min；
3. 4 DEG C of centrifugations of 12000rpm 15min prepare the EP pipes of corresponding labeled RNA printed words；
4. after centrifugation, solution is divided into three layers in EP pipes, and upper layer is the aqueous phase solution containing RNA, and centre is to contain genome
The batt layer of DNA, lower layer are red organic solution layer.
It is careful to take out about 500 μ L upper strata aqueous phase solution, move to the EP pipes of new labeled RNA printed words；
It is careful to take out about 300 μ L middle layer aqueous phase solutions, move to the EP pipes of new marker DNA printed words.
5. being separately added into 1 μ L RNasin and 500 μ L isopropanols in RNA EP pipes, mixing, 25 DEG C of standing 15min are overturned.
4 DEG C of 12000rpm 15min are centrifuged, at this time visible EP bottom of the tube white precipitate.It is careful that supernatant is sucked out, it discards.
RNA precipitate is washed 2 times with 1mL 75v/v% ethyl alcohol, i.e., is first incubated 3min, 4 DEG C of centrifugations of rear 12000rpm 5min.
After RNA precipitate washes, lies low to be positioned on toilet paper and dry (40~60min) afterwards with 30 μ L ddH2O dissolves, and 55
DEG C water-bath 30min, nano drop survey RNA concentration and purity.
6. 210 μ L of DNA precipitation reagents are added in DNA EP pipes, the DNA precipitation reagents are by 2 parts of 1,2-PD, ethyl alcohol 8
1.25 parts of preparations of 1.5M sodium acetates of part, pH=5~5.4 obtain.
It mixes well, cotton-shaped DNA precipitations visible at this time occur, 25 DEG C of standing 25min；
4 DEG C of 12000rpm 15min are centrifuged, at this time visible EP bottom of the tube white precipitate.It is careful that supernatant is sucked out, it discards.
DNA precipitations are washed once with absolute ethyl alcohol, and at 25 DEG C, 10000rpm centrifuges 12min；
After discarding supernatant, DNA precipitations continue to use the ethanol aqueous wash of 75v/v% primary, and the condition of centrifugation is
12000rpm, 5min, 4 DEG C or 25 DEG C.
The EP pipes of DNA, which lie low to be positioned on toilet paper, dries (40~60min) afterwards with 30 μ L TE buffer dissolvings, 55 DEG C
Water-bath 30min, Nano drop survey RNA concentration and purity.
Experimental example 1
DNA and RNA is carried out using the embodiment of the present invention 4 to 10 PBMC samples to extract, using Thermo companies
NanoDrop spectrophotometers distinguish the 10 PBMC cell DNAs and RNA concentration (ng/ μ L), purity (OD260/ of Detection and Extraction
OD280) and the degree that desalts (OD260/OD230) the results are shown in table below：
1 DNA testing results of table：(pure dna：OD260/OD280 ≈ 1.8, OD260/OD230 > 2.0)
2 RNA testing results of table：(pure rna：OD260/OD280 ≈ 2.0, OD260/OD230 > 2.0)
Experimental example 2
Comparative example is set on the basis of embodiment 4, and only extracts DNA, to evaluate DNA extraction effects.
1,2-PD in the DNA precipitation reagents is replaced with isometric 1,3-PD by comparative example 1；
1,2-PD in the DNA precipitation reagents is replaced with isometric ethyl alcohol by comparative example 2；
Sodium acetate in the DNA precipitation reagents is replaced with isometric ethyl alcohol by comparative example 3；
The DNA precipitation reagents are replaced with absolute ethyl alcohol by comparative example 4.
The DNA precipitation reagents are replaced with isopropanol by comparative example 5.
When experiment, first multigroup water in intermediate layer is mixed, then is packed as five parts, is precipitated respectively with five groups of DNA precipitation reagents,
To ensure that the consistency of experiment, experiment do 6 repetitions, compare average value.
Influence of the table 3 using different DNA precipitation reagents to DNA testing results
* p < 0.05, vs embodiment 4；* p < 0.01, vs embodiments 4.
Compared with Example 4, DNA concentration is declined comparative example 1, and OD260/OD280, OD260/OD230 do not conform to
Lattice.Although 1,3-PD compared with 1,2-PD, contains, there are two hydroxyls, and more symmetrical from the point of view of its molecular formula, pole
Property may smaller, but because it can form intramolecular hydrogen bond, two terminal hydroxy groups are easy to constitute six-membered ring structure, and possible reagent is striven
The ability for taking water by force is weaker, and DNA yields is caused to decline；Also it is not easy to be removed by ethyl alcohol after cyclization.
Compared with Example 4, DNA concentration is declined comparative example 2.Show that suitable 1,2- propylene glycol, which is added, effectively to be carried
High DNA yield.
Compared with Example 4, DNA concentration is declined comparative example 3.Show that suitable sodium acetate, which is added, to be effectively improved
DNA yields.
Compared with Example 4, DNA concentration declines to a great extent comparative example 4.Show that compound system of the present invention can be effective
Promote DNA yields.
Compared with Example 4, DNA concentration declines to a great extent comparative example 5, and OD260/OD280, OD260/OD230 do not conform to
Lattice.Show being not thorough for the removal of the phenol in system, either participates in many oil-soluble impurities or small molecule salt.Since PBMC comes
Derived from tissue samples, when extraction, may carry the substances such as some plasma compositions, under isopropanol may precipitate these impurity together
It splits.If the general this fields l Trizol+ isopropyl alcohol extractings, when subsequent wash, uses 0.1M sodium citrates, 10% ethyl alcohol to wash
Effect is better.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but it will be understood by those of ordinary skill in the art that：Its
It still can be with technical scheme described in the above embodiments is modified, either to which part or all technical features
Carry out equivalent replacement；And these modifications or replacements, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
Claims (1)
1. a kind of method for DNA and RNA in separation and Extraction cell, which is characterized in that including：
A) pyrolysis product is obtained using Trizol lytic cell samples release nucleic acid；
B) it is centrifuged after contacting the pyrolysis product at least one organic extraction solvent, so that the pyrolysis product is layered
To upper layer RNA water phases, the batt layer of middle level DNA and lower layer's organic phase；
C) batt layer of the RNA water phases and the DNA precipitated respectively, cleaned；
Wherein, when carrying out the precipitation, DNA precipitation reagents used include 1,2-PD；
Optionally, also include ethyl alcohol and/or the sodium acetate of pH=5~5.4 in the DNA precipitation reagents；
Optionally, the DNA precipitation reagents are mixed to get by the ingredient of following volumes part：
1~3 part of 1,2- propylene glycol, 6~10 parts of ethyl alcohol, pH=5~5.4 1~1.5 part of 1.5M sodium acetates；
Optionally, it when being precipitated to the batt layer of the DNA with the DNA precipitation reagents, first mixes well, then at 18 DEG C
15min~30min is stood at~30 DEG C；
Optionally, the volume ratio of the DNA precipitation reagents and the batt layer of the DNA is 0.6~0.8:0.8~1.2；
Optionally, when being cleaned to the DNA after precipitation, cleaning agent used is divided into the first cleaning agent and the second cleaning agent；
First cleaning agent is absolute ethyl alcohol；
Second cleaning agent is the ethanol water of 70v/v%~80v/v%；
Optionally, it when being cleaned to the DNA after precipitation with first cleaning agent or second cleaning agent, is precipitated with upper step
It is directly centrifuged after mixing；
Optionally, when being cleaned to the DNA after precipitation with first cleaning agent, it is described centrifugation at 18 DEG C~30 DEG C into
Row；
The condition of centrifugation is 9000rpm~11000rpm, 10min~15min；
Optionally, when being cleaned to the DNA after precipitation with second cleaning agent, the condition of centrifugation be 11000rpm~
13000rpm, 3min~7min；
Optionally, the RNA precipitate reagent includes isopropanol；
Optionally, the RNA precipitate reagent further includes that RNase inhibitor is appropriate；
Optionally, the RNase inhibitor be selected from 8-hydroxyquinoline, guanidinium isothiocyanate, vanadyl-ribonucleoside complex,
It is one or more in RNasin, SDS, beta -mercaptoethanol；
Optionally, when being precipitated to the RNA water phases with the RNA precipitate reagent, at 18 DEG C~30 DEG C after mixing well
Lower standing 10min~20min；
Optionally, the volume ratio of the RNA precipitate reagent and the RNA water phases is 0.8~1.2:0.8~1.2；
Optionally, when being cleaned to the RNA after precipitation, cleaning agent used is that the ethyl alcohol of 70v/v%~80v/v% is water-soluble
Liquid；
Optionally, when being cleaned to the RNA after precipitation with the cleaning agent, with 2 DEG C~6 DEG C incubations after upper step precipitation mixing
1min~5min, centrifugation；
The condition of centrifugation is 2 DEG C~6 DEG C, 11000rpm~13000rpm, 3min~7min；
Optionally, when cracking PBMC samples using Trizol, linearisation acrylamide is also contained in cracking system；
Optionally, the condition of the cracking is：
Trizol reagents and the cell sample mixing are first used, linearisation acrylamide is added after standing 15min~25min,
3min~8min is stood after mixing；
Optionally, the organic extraction solvent is selected from chloroform or the bromo- 3- chloropropanes of 1-；
Optionally, in step a), the centrifugation is under conditions of 2 DEG C~6 DEG C, 10000rpm~14000rpm centrifuges
10min~20min；
Optionally, the cell sample is zooblast sample；
Optionally, the zooblast sample is mammalian cell sample；
Optionally, the mammalian cell sample is primates zooblast sample；
Optionally, the primates zooblast sample is the cell sample of people；
Optionally, the cell sample of the people is PBMC samples；
Optionally, the PBMC samples pass through Ficoll-Hypaque density-gradient centrifugation methods, Percoll density gradient centrifugations
Any one of method, Iodixanol gradient centrifugations, hydroxyethyl starch (HES) centrifugal method is isolated.
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| CN119265282A (en) * | 2024-11-26 | 2025-01-07 | 上海体育大学 | Dried blood spot pretreatment method and RNA extraction method |
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Application publication date: 20181113 |