CN116676303A - Soil RNA extraction kit free of water saturated phenol and chloroform, extraction method and application thereof - Google Patents
Soil RNA extraction kit free of water saturated phenol and chloroform, extraction method and application thereof Download PDFInfo
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 title claims abstract description 65
- 239000002689 soil Substances 0.000 title claims abstract description 65
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 55
- 238000010802 RNA extraction kit Methods 0.000 title claims abstract description 31
- 150000002989 phenols Chemical class 0.000 title claims abstract description 25
- 238000000605 extraction Methods 0.000 title abstract description 16
- 230000004568 DNA-binding Effects 0.000 claims abstract description 29
- 239000006166 lysate Substances 0.000 claims abstract description 19
- 239000000243 solution Substances 0.000 claims description 49
- 238000000034 method Methods 0.000 claims description 26
- 230000004570 RNA-binding Effects 0.000 claims description 23
- 238000004140 cleaning Methods 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims description 16
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 claims description 14
- 239000004021 humic acid Substances 0.000 claims description 14
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 11
- 239000002516 radical scavenger Substances 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 8
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 8
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 8
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 8
- 239000008118 PEG 6000 Substances 0.000 claims description 6
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 6
- 239000007983 Tris buffer Substances 0.000 claims description 6
- 239000011324 bead Substances 0.000 claims description 6
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 6
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- 239000011259 mixed solution Substances 0.000 claims description 6
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- 238000005406 washing Methods 0.000 claims description 6
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- 230000010355 oscillation Effects 0.000 claims description 4
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- 238000011084 recovery Methods 0.000 claims description 2
- 108020004707 nucleic acids Proteins 0.000 abstract description 16
- 150000007523 nucleic acids Chemical class 0.000 abstract description 16
- 102000039446 nucleic acids Human genes 0.000 abstract description 16
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 12
- 238000002123 RNA extraction Methods 0.000 abstract description 11
- 239000000126 substance Substances 0.000 abstract description 9
- 231100000331 toxic Toxicity 0.000 abstract description 4
- 230000002588 toxic effect Effects 0.000 abstract description 4
- 238000005336 cracking Methods 0.000 abstract description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 17
- 102000004169 proteins and genes Human genes 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 102000006382 Ribonucleases Human genes 0.000 description 6
- 108010083644 Ribonucleases Proteins 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- 239000011734 sodium Substances 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 150000004676 glycans Chemical class 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 4
- 238000002205 phenol-chloroform extraction Methods 0.000 description 4
- 229920001282 polysaccharide Polymers 0.000 description 4
- 239000005017 polysaccharide Substances 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 239000008346 aqueous phase Substances 0.000 description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 102000011931 Nucleoproteins Human genes 0.000 description 2
- 108010061100 Nucleoproteins Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000002934 lysing effect Effects 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- -1 salt ions Chemical class 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- OCKGFTQIICXDQW-ZEQRLZLVSA-N 5-[(1r)-1-hydroxy-2-[4-[(2r)-2-hydroxy-2-(4-methyl-1-oxo-3h-2-benzofuran-5-yl)ethyl]piperazin-1-yl]ethyl]-4-methyl-3h-2-benzofuran-1-one Chemical compound C1=C2C(=O)OCC2=C(C)C([C@@H](O)CN2CCN(CC2)C[C@H](O)C2=CC=C3C(=O)OCC3=C2C)=C1 OCKGFTQIICXDQW-ZEQRLZLVSA-N 0.000 description 1
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 1
- 235000017491 Bambusa tulda Nutrition 0.000 description 1
- 241001330002 Bambuseae Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- SWLVFNYSXGMGBS-UHFFFAOYSA-N ammonium bromide Chemical compound [NH4+].[Br-] SWLVFNYSXGMGBS-UHFFFAOYSA-N 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 239000011425 bamboo Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- RLGQACBPNDBWTB-UHFFFAOYSA-N cetyltrimethylammonium ion Chemical compound CCCCCCCCCCCCCCCC[N+](C)(C)C RLGQACBPNDBWTB-UHFFFAOYSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- BKHZIBWEHPHYAI-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol Chemical compound ClC(Cl)Cl.CC(C)CCO BKHZIBWEHPHYAI-UHFFFAOYSA-N 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000005431 greenhouse gas Substances 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002563 ionic surfactant Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002366 mineral element Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000006228 supernatant Substances 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
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- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
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Abstract
The invention discloses a soil RNA extraction kit without water saturated phenol and chloroform, and an extraction method and application thereof. The kit comprises a lysate and a DNA binding column; the lysate contains 2% -10% (w/v) SDS, 0.2-0.5M Na 2 HPO 4 Ph=7.5 to 8.5. The invention adopts SDS and DNA binding column to replace the traditional phenol and chloroform, the SDS is used for cracking cells and releasing nucleic acid, and then the pollution of genome DNA is removed by means of DNA collecting column, thereby achieving the purpose of further purifying RNA. The soil RNA extraction kit and the extraction method provided by the invention do not contain toxic and harmful substances such as water saturated phenol, chloroform and the like, have higher safety, and have good RNA extraction effect.
Description
Technical Field
The invention relates to the technical field of soil RNA extraction, in particular to a soil RNA extraction kit without water saturated phenol and chloroform, and an extraction method and application thereof.
Background
The soil contains various microorganisms which play an important role in the circulation of soil organic matters and mineral elements, and regulate and control various ecological processes such as the generation and emission of greenhouse gases such as methane and the like. Research shows that the higher the microbial diversity in the soil, the more ecological functions and the higher environmental stress resistance and crop productivity are achieved. Soil is an open ecological system, and the distribution and functions of the soil microorganism diversity can be affected by the changes of environmental factors such as illumination, temperature, soil pH value and the like. The response of soil microorganisms to the change of the external environment can be timely found through the research on RNA.
Although various methods have been developed for extraction and purification of RNA in soil, including (1) Trizol reagent rapid extraction, trizol is a novel total RNA extraction reagent whose main components are phenol, guanidine isothiocyanate, sarcosyl and the like, wherein phenol has a main role of cell lysis, guanidine isothiocyanate is a strong denaturing reagent which not only inhibits RNase activity but also effectively separates nucleic acids from nucleoprotein. Trizol reagent is suitable for rapid isolation of total RNA from various tissues or cells of human, animal, plant, microorganism, etc. (2) The CTAB reagent method, CTAB is known collectively as cetyltrimethylammonium bromide (Cetyltrimethylammonium Ammonium Bromide), which is one of the cationic detergents, has the property of precipitating the polysaccharide with nucleic acid from a low ionic strength solution, while in a high ionic strength solution, it forms a complex with the protein and polysaccharide. By utilizing the characteristic, the nucleic acid can be separated from the protein and the polysaccharide, and impurities such as the protein, the saccharide, the phenols and the like are removed again through extraction of chloroform isoamyl alcohol. (3) SDS-phenol method, sodium Dodecyl Sulfate (SDS) is an anionic surfactant, has the function of denaturing protein, is used for destroying cell wall and separating nucleoprotein from nucleic acid in nucleic acid extraction operation, is often used in proportion with beta-mercaptoethanol, phenol and the like, beta-mercaptoethanol is usually used for reducing disulfide bond, is used for inhibiting RNase activity in nucleic acid extraction, phenol has the characteristic of rapid protein denaturation, and water saturated phenol (also called acidic phenol) is usually used for RNA extraction because RNA is unstable and is easy to degrade in alkaline environment. Phenol can denature proteins rapidly, but does not completely inhibit the activity of RNase, phenol can be used: chloroform: isoamyl alcohol (25:24:1) is effective in removing proteins, lipids, polysaccharides, and some cell debris. The SDS-phenol method can inhibit RNase and denature proteins, and can effectively crack soil samples by combining a glass bead physical crushing method so as to extract a large amount of total RNA of the soil. Therefore, the soil RAN extraction method in the prior art is basically based on the water saturated phenol and chloroform extraction mode to achieve the purposes of cell lysis and humic acid removal. Water saturated phenol and chloroform are organic chemical substances harmful to human bodies and the environment, and meanwhile, the chloroform is used as an easy-to-poison control chemical, and the circulation and the use of the chloroform are limited.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings in the prior art and provide a soil RNA extraction kit without water saturated phenol and chloroform.
The second object of the present invention is to provide a method for extracting RNA from soil free of water-saturated phenol and chloroform.
A third object of the present invention is to provide the use of the soil RNA extraction kit and method free of water saturated phenol and chloroform.
The above object of the present invention is achieved by the following technical solutions:
in existing conventional RNA extraction protocols, the lysate typically contains water saturated phenol and chloroform. Both can denature protein and inhibit the activity of RNase; in addition, chloroform can also remove phenol and some fat-soluble substances from the aqueous phase. Phenol was used: chloroform: the upper layer after isoamyl alcohol extraction and centrifugation is an aqueous phase containing RNA, the middle is denatured protein and DNA, and the lower layer is an organic solvent phase, so that the upper aqueous phase is separated, and the effect of RNA purification is achieved. As described in the background art, both water-saturated phenol and chloroform are organic chemicals harmful to human body and environment, and at the same time, chloroform is used as an easy-to-poison controlling chemical, and its circulation and use are limited. In order to solve the problems, the invention aims to provide a soil RNA extraction kit and a method without water saturated phenol and chloroform.
The invention firstly provides a soil RNA extraction kit without water saturated phenol and chloroform, which comprises a lysate and a DNA binding column; the lysate contains 2% -10% (w/v) SDS, 0.2-0.5M Na 2 HPO 4 The balance being water, ph=7.5 to 8.5.
The present invention uses SDS and DNA binding column to replace traditional phenol and chloroform. Wherein SDS is an ionic surfactant,which can bind to proteins as R-O-SO 3 - … r+ -protein complex, which denatures the protein and precipitates. Thus, SDS can solubilize cell membranes, depolymerize nuclear proteins in cells, inhibit the action of ribonucleases, and promote separation of proteins from nucleic acids. Thus, in the present invention, SDS was used to lyse cells and release nucleic acid, and phosphate was used to maintain the reaction system in a relatively stable pH environment, and at the same time, to reduce the adsorption of nucleic acid by soil and thus to increase the yield of nucleic acid. As the supernatant after cleavage and centrifugation contains both genomic DNA pollution and target product RNA, the genomic DNA pollution is removed by using a DNA collection column (namely, a DNA binding column is used as a impurity removal tool), so that the aim of further purifying RNA is fulfilled. The lysate component of the soil RNA extraction kit provided by the invention does not contain toxic and harmful substances such as water saturated phenol, chloroform and the like, has higher safety, and has good RNA extraction effect.
Preferably, the lysate comprises 5% -10% (w/v) SDS, 0.3-0.5M Na 2 HPO 4 The balance water, ph=8.0.
Preferably, the DNA binding column is DNA Mini Columns|Omega Bio-tek。
Further, the soil RNA extraction kit also comprises a humic acid scavenger, wherein the humic acid scavenger is 1% -10% (w/v) polyvinylpyrrolidone water solution, and is used for removing humic acid in soil.
Preferably, the humic acid scavenger is 5% -10% (w/v) polyvinylpyrrolidone aqueous solution.
Further, the soil RNA extraction kit also comprises a DNA binding solution, wherein the DNA binding solution contains 50% (w/v) guanidine isothiocyanate, 0.5-1.5M KCl and 0.1-1M NH 4 Cl, 0.1-0.3M NaAc, and the balance of water. The DNA binding solution has the function of promoting the binding of DNA and DNA binding columns, so that the genomic DNA is better adsorbed and the genomic DNA pollution is better removed.
PreferablyThe DNA binding solution contains 50% (w/v) guanidine isothiocyanate, 1M KCl,0.5M NH 4 Cl,0.1M NaAc, balance water.
Further, the soil RNA extraction kit also comprises an RNA binding solution, wherein the RNA binding solution contains 1.0-3.0M NaCl, 10-30% (w/v) PEG6000, 30-50% (v/v) isopropanol and the balance of water. The function of the RNA binding solution is to promote the binding of RNA and the RNA binding column, so that the target product RNA is better adsorbed.
Preferably, the RNA binding solution contains 1.0M NaCl,10% (w/v) PEG6000, 30% (v/v) isopropanol, and the balance water.
Further, the soil RNA extraction kit also comprises an RNA cleaning solution I, wherein the RNA cleaning solution I contains 10% -30% (v/v) ethanol, 10mM Tris, 0.5-1M guanidine isothiocyanate and the balance of water. The RNA cleaning solution I has the function of removing impurities such as proteins, wherein the guanidine isothiocyanate has the function of promoting the combination of nucleic acid and a silica gel column.
Preferably, the RNA washing solution I contains 20% (v/v) ethanol, 10mM Tris,1M guanidine isothiocyanate, the balance being water.
Further, the soil RNA extraction kit also comprises an RNA cleaning solution II, wherein the RNA cleaning solution II is 70% (v/v) ethanol water solution. The effect of the RNA cleaning solution II is to remove salt ions.
The invention also provides application of any soil RNA extraction kit in extracting soil RNA.
The invention also provides a method for extracting soil RNA by adopting any one of the soil RNA extraction kits, which comprises the following steps:
s1, mixing a soil sample with glass beads and a lysate, and carrying out vortex oscillation for 8-12 min; then centrifugally separating at the temperature of 0-4 ℃ and the speed of 4000-10000 g for 5-10 min;
s2, taking the upper clear liquid after the centrifugation in the step S1, adding 0.1 times of the volume of humic acid scavenger and the equal volume of DNA binding solution, shaking and mixing uniformly, and centrifuging 10000-12000 g for 2-4 min at the temperature of 0-4 ℃;
s3, transferring the upper clear liquid after the centrifugation in the step S2 into a DNA binding column nested in a centrifuge tube, centrifugally separating 10000-12000 g for 1-2 min, and discarding the column;
s4, adding the sample centrifuged in the step S3 into an equal volume of RNA binding solution, and uniformly oscillating and mixing;
s5, transferring the sample mixed solution obtained in the step S4 into an RNA binding column nested in a centrifuge tube, centrifugally separating 10000-12000 g for 1-2 min, and discarding waste liquid; repeating for a plurality of times until the sample mixed solution in the step S4 is completely transferred through the column;
s6, sleeving the RNA binding column in the step S5 into a new collecting pipe, adding an RNA cleaning solution I, centrifuging 10000-12000 g for 1-2 min, and discarding the filtering;
s7, adding an RNA cleaning solution II into the RNA combined column sleeve recovery header of the step S6, centrifuging 10000-12000 g for 1-2 min, and discarding the filtrate; repeating the steps;
s8, recovering the RNA combined column sleeve in the step S7 into a collecting pipe, and centrifuging 10000-12000 g for 2-4 min;
s9, sleeving the RNA binding column in the step S8 into a new collecting pipe, adding DEPC (DEPC) treated water, standing at room temperature, centrifuging 10000-12000 g for 1-2 min, and eluting RNA to obtain the RNA.
The SDS in the lysate adopted in the step S1 is used for lysing cells and releasing nucleic acid, so that fatty acid, lipid and protein on cell membranes can be destroyed; by combining with the glass bead breaking method, cells can be broken to the maximum extent, and target RNA with high yield can be obtained.
Further, the DNA binding column is HiBind DNA Mini Column, i.eDNA collection column in DNA Mini columns|omega Bio-tek kit.
Further, the RNA binding Column is HiBind RNA Mini Column, and the RNA collection Column in the HiBind@RNA Mini column|omega Bio-tek kit.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a soil RNA extraction kit without water saturated phenol and chloroform, and an extraction method and application thereof. The kit comprises a lysate and a DNA binding column; the lysate only contains the lysis active ingredient SDS. The invention adopts SDS and DNA binding column to replace the traditional phenol and chloroform, the SDS is used for cracking cells and releasing nucleic acid, and then the pollution of genome DNA is removed by means of DNA collecting column, thereby achieving the purpose of further purifying RNA. The soil RNA extraction kit and the extraction method provided by the invention do not contain toxic and harmful substances such as water saturated phenol, chloroform and the like, have higher safety, and have better RNA extraction effect compared with the traditional phenol-chloroform extraction method. The soil RNA extraction kit and the method are suitable for extracting RNA in different types of soil, and have wide application range.
Drawings
FIG. 1 is a graph showing RNA extraction effect of the present invention on different soil types.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
DNA Mini Columns|Omega Bio-tek
HiBind@RNA Mini Column|Omega Bio-tek
Example 1A soil microbial RNA extraction kit free of Water saturated phenol and chloroform
A soil microbial RNA extraction kit free of water saturated phenol and chloroform, comprising:
(1) Lysates and DNA binding columns; the lysate contains 2% -10% (w/v) SDS, 0.2-0.5M Na 2 HPO, balance water, ph=8.0; the preparation method of the lysate comprises the following steps: SDS and Na were weighed according to the formula 2 HPO 4 Dissolving in water, and adjusting pH to 8.0.
Wherein SDS is used for lysing cells and releasing nucleic acid, and is removed by means of a DNA collecting columnAnd the genome DNA pollution is removed, so that the aim of further purifying RNA is achieved, and the traditional phenol and chloroform functions are replaced. The DNA binding column is DNA Mini Columns|Omega Bio-tek。
(2) Humic acid scavenger: an aqueous solution containing 1 to 10% (w/v) polyvinylpyrrolidone; the configuration method comprises the following steps: weighing polyvinylpyrrolidone solid according to a formula, dissolving the polyvinylpyrrolidone solid in water, and fixing the volume. The humic acid scavenger is used for removing humic acid.
(3) DNA binding solution: contains 50% (w/v) guanidine isothiocyanate, 0.5-1.5M KCl, 0.1-1 MNH 4 Cl, 0.1-0.3M NaAc, and the balance of water. The configuration method comprises the following steps: according to the formula, the solid of each component is weighed and dissolved in water to be fixed in volume. The DNA binding solution is used for promoting the combination of DNA and DNA binding columns, so that the genomic DNA is better adsorbed and the genomic DNA pollution is better removed.
(4) RNA binding solution: contains 1.0-3.0M NaCl, 10-30% (w/v) PEG6000, 30-50% (v/v) isopropanol and water. The configuration method comprises the following steps: according to the formula, weighing solid, measuring liquid, adding water and fixing volume. The function of the RNA binding solution is to promote the binding of RNA and the RNA binding column, so that the target product RNA is better adsorbed.
(5) RNA cleaning solution I: the composition contains 10-30% (v/v) ethanol, 10mM Tris, 0.5-1M guanidine isothiocyanate and the balance of water. The configuration method comprises the following steps: according to the formula, weighing solid, measuring liquid, adding water and fixing volume. The RNA cleaning solution I has the function of removing impurities such as proteins, wherein the guanidine isothiocyanate has the function of promoting the combination of nucleic acid and a silica gel column.
(6) RNA washing solution II:70% (v/v) aqueous ethanol. The configuration method comprises the following steps: according to the formula, liquid is measured, and water is added to fix volume. The function of the RNA cleaning fluid II is to remove salt ions.
Example 2A method for extracting RNA from soil microorganisms free of aqueous saturated phenol and chloroform
(1) 0.5g of soil sample (No. 1 soil, bamboo forest soil and river mud) is weighed respectively and added into a 10mL centrifuge tube,0.5g of glass beads was added, and 700. Mu.L of lysate (10% w/v SDS,0.3M Na) 2 HPO 4 Water in balance, ph=8.0);
(2) Placing the sample in the step 1 into a vortex mixer, and carrying out vortex oscillation at the maximum rotation speed for 10min;
(3) Centrifuging the sample in step 2 at 4deg.C under a centrifugal force of 4000g (10000 g) for 10min (5 min);
(4) Transfer 400. Mu.L of the upper clear liquid of the sample from step 3 to a 2mL centrifuge tube, add 0.1 volumes of humic acid scavenger (5% w/v aqueous polyvinylpyrrolidone) and equal volumes of DNA binding solution (containing 50% w/v guanidine isothiocyanate, 1M KCl,0.5M NH) 4 Cl,0.1M NaAc, the balance of water) and uniformly mixing by shaking;
(5) Centrifuging the sample obtained in the step 4 at a temperature of 4 ℃ and a centrifugal force of 12000g for 2min;
(6) Transferring the upper clear liquid of the sample in the step 5 into a HiBind DNA Mini Column nested in a 2mL centrifuge tube, centrifugally separating for 1min under the centrifugal force of 12000g, and discarding the column;
(7) Adding the sample obtained in the step 6 into an equal volume of RNA binding solution (1M NaCl,10% w/v PEG6000, 30% v/v isopropanol and the balance being water), and shaking and uniformly mixing;
(8) Transferring the sample in the step 7 into a HiBind RNA Mini Column nested in a 2mL centrifuge tube, centrifugally separating for 1min under the centrifugal force of 12000g, and discarding the waste liquid;
(9) Repeating the step 8 until the mixed solution in the step 7 is completely transferred through the column;
(10) The HiBind RNA Mini Column of step 9 was sleeved into a new 2mL collection tube, 500. Mu.L of RNA washing solution I (20% v/v ethanol, 10mM Tris,1M guanidine isothiocyanate, the balance being water) was added, centrifuged at 12,000g for 1min, and the filtration was discarded;
(11) The HiBind RNA Mini Column in the step 10 is put back into a 2mL collecting pipe, 700 mu L of RNA cleaning liquid II (70% v/v ethanol water solution) is added, and the mixture is centrifuged for 1min at 12,000g and is discarded;
(12) Repeating step 11;
(13) The HiBind RNA Mini Column in the step 12 is sleeved back into a 2mL collecting pipe, and the centrifugation is carried out for 2min at 12,000 g;
(14) Sleeving HiBind RNA Mini Column in the step 13 into a new 1.5mL collecting pipe, adding 30-50 mu L DEPC treated water, standing at room temperature for 1min, centrifuging at 12,000g for 1min, and eluting RNA;
(15) The sample RNA in step 14 is stored at-80℃to facilitate subsequent analysis.
Example 3A method for extracting RNA from soil microorganisms free of aqueous saturated phenol and chloroform
(1) 0.5g of soil samples (garden soil 1, garden soil 2, garden soil) were weighed into 10mL centrifuge tubes, 0.5g of glass beads were added, and 700. Mu.L of lysate (5% w/v SDS,0.5M Na) 2 HPO 4 Water in balance, ph=8.0);
(2) Placing the sample in the step 1 into a vortex mixer, and carrying out vortex oscillation at the maximum rotation speed for 10min;
(3) Centrifuging the sample in step 2 at 4deg.C under a centrifugal force of 4000g (10000 g) for 10min (5 min);
(4) Transfer 400. Mu.L of the upper clear liquid of the sample from step 3 to a 2mL centrifuge tube, add 0.1 volumes of humic acid scavenger (10% w/v aqueous polyvinylpyrrolidone) and equal volumes of DNA binding solution (containing 50% guanidine isothiocyanate, 1M KCl,0.5M NH) 4 Cl,0.1M NaAc, the balance of water) and uniformly mixing by shaking;
(5) Centrifuging the sample obtained in the step 4 at a temperature of 4 ℃ and a centrifugal force of 12000g for 2min;
(6) Transferring the upper clear liquid of the sample in the step 5 into a HiBind DNA Mini Column nested in a 2mL centrifuge tube, centrifugally separating for 1min under the centrifugal force of 12000g, and discarding the column;
(7) Adding the sample obtained in the step 6 into an equal volume of RNA binding solution (1M NaCl,10% w/v PEG6000, 30% v/v isopropanol and the balance being water), and shaking and uniformly mixing;
(8) Transferring the sample in the step 7 into a HiBind RNA Mini Column nested in a 2mL centrifuge tube, centrifugally separating for 1min under the centrifugal force of 12000g, and discarding the waste liquid;
(9) Repeating the step 8 until the mixed solution in the step 7 is completely transferred through the column;
(10) The HiBind RNA Mini Column of step 9 was sleeved into a new 2mL collection tube, 500. Mu.L of RNA washing solution I (20% v/v ethanol, 10mM Tris,1M guanidine isothiocyanate, the balance being water) was added, centrifuged at 12,000g for 1min, and the filtration was discarded;
(11) The HiBind RNA Mini Column in the step 10 is put back into a 2mL collecting pipe, 700 mu L of RNA cleaning liquid II (70% v/v ethanol water solution) is added, and the mixture is centrifuged for 1min at 12,000g and is discarded;
(12) Repeating step 11;
(13) The HiBind RNA Mini Column in the step 12 is sleeved back into a 2mL collecting pipe, and the centrifugation is carried out for 2min at 12,000 g;
(14) Sleeving HiBind RNA Mini Column in the step 13 into a new 1.5mL collecting pipe, adding 30-50 mu L DEPC treated water, standing at room temperature for 1min, centrifuging at 12,000g for 1min, and eluting RNA;
(15) The sample RNA in step 14 is stored at-80℃to facilitate subsequent analysis.
Examples 2 and 3 RNA extraction Effect against different soil types the graph is shown in FIG. 1, the electrophoresis bands show RNA band integrity, OD 260 /OD 280 About 1.8-2.0 OD 260 /OD 230 About 0.9-2.1 OD 260 /OD 280 The ratio is better than that of the phenol chloroform extraction method, and the OD 260 /OD 230 The result of the phenol chloroform extraction method is almost the same.
The results show that the soil RNA extraction kit and the extraction method provided by the invention do not contain toxic and harmful substances such as water saturated phenol, chloroform and the like, are higher in safety, have better RNA extraction effect compared with the traditional phenol-chloroform extraction method, and are suitable for extracting RNA in different types of soil, and are wide in application range.
Claims (10)
1. The soil RNA extraction kit without water saturated phenol and chloroform is characterized by comprising a lysate and a DNA binding column; the lysate contains 2% -10% (w/v) SDS, 0.2-0.5M Na 2 HPO 4 The balance being water, ph=7.5 to 8.5.
2. The soil RNA extraction kit of claim 1, further comprising a humic acid scavenger, wherein the humic acid scavenger is 1% to 10% w/v polyvinylpyrrolidone aqueous solution.
3. The soil RNA extraction kit of claim 1, further comprising a DNA binding solution comprising 50% w/v guanidine isothiocyanate, 0.5-1.5M KCl, 0.1-1 MNH 4 Cl, 0.1-0.3M NaAc, and the balance of water.
4. The soil RNA extraction kit of claim 1, further comprising an RNA binding solution comprising 1.0-3.0 m nacl, 10-30% w/v PEG6000, 30-50% v/v isopropanol, the balance being water.
5. The soil RNA extraction kit of claim 1, further comprising an RNA cleaning solution I, wherein the RNA cleaning solution I contains 10% -30% v/v ethanol, 10mM Tris, 0.5-1M guanidine isothiocyanate and the balance water.
6. The soil RNA extraction kit of claim 1, further comprising an RNA washing solution II, wherein the RNA washing solution II is 70% v/v ethanol aqueous solution.
7. Use of the soil RNA extraction kit of any one of claims 1 to 6 for extracting soil RNA.
8. A method for extracting soil RNA using the soil RNA extraction kit of any one of claims 1 to 6, comprising the steps of:
s1, mixing a soil sample with glass beads and a lysate, and carrying out vortex oscillation for 8-12 min; then centrifugally separating at the temperature of 0-4 ℃ and the speed of 4000-10000 g for 5-10 min;
s2, taking the upper clear liquid after the centrifugation in the step S1, adding 0.1 times of the volume of humic acid scavenger and the equal volume of DNA binding solution, shaking and mixing uniformly, and centrifuging 10000-12000 g for 2-4 min at the temperature of 0-4 ℃;
s3, transferring the upper clear liquid after the centrifugation in the step S2 into a DNA binding column nested in a centrifuge tube, centrifugally separating 10000-12000 g for 1-2 min, and discarding the column;
s4, adding the sample centrifuged in the step S3 into an equal volume of RNA binding solution, and uniformly oscillating and mixing;
s5, transferring the sample mixed solution obtained in the step S4 into an RNA binding column nested in a centrifuge tube, centrifugally separating 10000-12000 g for 1-2 min, and discarding waste liquid; repeating for a plurality of times until the sample mixed solution in the step S4 is completely transferred through the column;
s6, sleeving the RNA binding column in the step S5 into a new collecting pipe, adding an RNA cleaning solution I, centrifuging 10000-12000 g for 1-2 min, and discarding the filtering;
s7, adding an RNA cleaning solution II into the RNA combined column sleeve recovery header of the step S6, centrifuging 10000-12000 g for 1-2 min, and discarding the filtrate; repeating the steps;
s8, recovering the RNA combined column sleeve in the step S7 into a collecting pipe, and centrifuging 10000-12000 g for 2-4 min;
s9, sleeving the RNA binding column in the step S8 into a new collecting pipe, adding DEPC (DEPC) treated water, standing at room temperature, centrifuging 10000-12000 g for 1-2 min, and eluting RNA to obtain the RNA.
9. The method of claim 8, wherein the DNA binding column is HiBind DNA Mini Column.
10. The method of claim 8, wherein the RNA binding column is HiBind RNA Mini Column.
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| CN202310865434.7A CN116676303A (en) | 2023-07-13 | 2023-07-13 | Soil RNA extraction kit free of water saturated phenol and chloroform, extraction method and application thereof |
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