CN101935648A - Method and kit for extracting ribonucleic acid (RNA) - Google Patents
Method and kit for extracting ribonucleic acid (RNA) Download PDFInfo
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- CN101935648A CN101935648A CN 201010281633 CN201010281633A CN101935648A CN 101935648 A CN101935648 A CN 101935648A CN 201010281633 CN201010281633 CN 201010281633 CN 201010281633 A CN201010281633 A CN 201010281633A CN 101935648 A CN101935648 A CN 101935648A
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Abstract
The invention relates to the field of molecular biology and discloses a method and a kit for extracting ribonucleic acid (RNA). The method for extracting the RNA comprises the following steps of: uniformly mixing a sample and lysis solution; centrifugally collecting supernate and uniformly mixing the supernate and absolute ethyl alcohol which is half of the volume of the supernate; combining the mixture with 0.45 mu m glass fiber cellulose acetate membrane; washing with deproteinization liquid and rinsing liquid; eluting adsorbed RNA; uniformly mixing lysis cells and an extraction aid; and centrifugally removing polysaccharide and polyphenol substances. The kit for extracting the RNA comprises the lysis solution, the deproteinization liquid, the rinsing liquid, the eluting liquid and the extraction aid. The method of the invention has the advantages of simpleness, rapidness, no use of toxic reagent, wide application range and capabilities of effectively removing the polysaccharide and polyphenol substances and separating high-quality RNA out by adding the extraction aid. The kit of the invention has the advantages of no toxic reagent, wide application range, RNA extraction effects of plant materials which are rich in polysaccharide and polyphenol superior to that of foreign kits, low cost and suitability for extensive laboratories and scientific researches.
Description
Technical field
The present invention relates to biology field, relate to method and the test kit of a kind of RNA of extraction specifically.
Background technology
Extracting RNA from tissue is the prerequisite of carrying out the research of molecular biology aspect, and the good RNA of dna purity height, integrity has great importance for researchs such as the structure in Northern hybridization analysis, mRNA separation, external translation, cDNA library, RT-PCR isolated genes, difference display analysis.Many materials make that effective separation and purification of these materials RNA is extremely difficult, thereby have hindered its molecular biology aspect progress of research owing to be rich in phenolic compound and polysaccharide and contain the secondary metabolite that some still can't be determined.
The extracting method of RNA commonly used has the total RNA extraction reagent box of TRIZOL method, guanidine isothiocyanate method, CTAB-LiCl method and domestic and international each biotech firm etc.The classical way TRIZOL method that RNA extracts, it is a kind of method based on guanidinium isothiocyanate/phenol/chloroform extracting principle, this method is applied widely, animals and plants all are suitable for, the RNA that extracts does not have the pollution of genomic dna substantially, but this method complex operation step extracts in the reagent and used toxic reagents such as phenol, chloroform, and this method is not suitable for the extraction of being rich in polysaccharide polyphenol material RNA.The RNeasy Plant Mini Kit of the external well-known Qiagen of biotech firm is a kind of test kit that carries out the RNA extraction specially at vegetable material, the method that this test kit adopts is based on guanidinium isothiocyanate cracked principle, this test kit all is suitable for most of vegetable material, it is fast to have extraction rate, the purity height, do not use advantages such as toxic reagent, but it is undesirable at animal material, microbial material RNA extraction effect, especially be rich in the material of polysaccharide, because polysaccharide is not easy to separate with RNA and is easy to adsorption column is stopped up, thus the failure that causes RNA to extract.The CTAB-LiCl method often is used to extract the extraction of being rich in polysaccharide polyphenol material RNA, but since CTAB itself be one with the similar reagent of SDS function, although it can form insoluble mixture with RNA and DNA, help removing polysaccharide, but the CTAB method will be operated in conjunction with other usually, through steps such as LiCl precipitations, make this method complex operation as again, and when extracting, use the toxic reagent chloroform.
Summary of the invention
The object of the invention provides a kind of simple and fast, does not use toxic reagent, can extract the method for high-quality RNA from multiple materials such as animal, plant and microorganism.
For realizing purpose of the present invention, the present invention adopts following technical scheme:
A kind of method of extracting RNA comprises:
Step 1: sample and lysate mixing, centrifugal, it is 10～50mmol/L that described lysate contains concentration, the Tris-HCl of pH value 6.5～8.0,3～5mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol;
Step 2: get step 1 gained supernatant,, combine with the plain film of 0.45 μ m glass fibre with the dehydrated alcohol mixing of 1/2 volume RNase-free;
Step 3: wash the plain film of described glass fibre with protein liquid removal and rinsing liquid, it is 5～50mmol/L that described protein liquid removal contains concentration, the Tris-HCl of pH value 6.5～8.0,0.1～0.5mol/L guanidinium isothiocyanate and 5%～20% dehydrated alcohol, it is 5～50mmol/L that described rinsing liquid contains concentration, the Tris-HCl of pH value 6.5～8.0,50～200mmol/LNaCl and 50%～80% dehydrated alcohol;
Step 4: the RNA that adsorbs on the plain film of the described glass fibre of wash-out.
The method of extraction RNA of the present invention, step 1 lysate and sample mixing, for the extraction of RNA provides optimum buffer condition, it is 10～50mmol/L that described lysate contains concentration, the Tris-HCl of pH value 6.5～8.0,3～5mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol, do not contain poisonous reagent benzene phenol, activity that can strongly inhibited RNase, efficiently lysing cell discharges RNA again.Guanidinium isothiocyanate belongs to separates even agent, is the protein denaturant of a class brute force, and solubilized protein also makes secondary protein structure disappear, and causes the cellularstructure degraded, the rapid and separate nucleic acid of nucleoprotein.
Wherein, as preferably, it is 25mmol/L that described lysate contains concentration, the Tris-HCl of pH value 7.5,4mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol.
The method of extraction RNA of the present invention, combine with the plain film of 0.45 μ m glass fibre behind the dehydrated alcohol mixing of step 1 gained supernatant and 1/2 volume RNase-free, ethanol can make RNA by on the plain film of the described glass fibre of being combined in of selectivity, and metabolism product debonds such as albumen, genomic dna and other polysaccharide polyphenols are on the plain film of described glass fibre.
Protein is an important factor polluting the RNA sample, obtain complete, high-quality RNA and just must remove albumen impurity effectively.Ordinary method mostly adopts phenol, chloroform extracting to remove protein.The method of extraction RNA of the present invention, step 3 utilizes protein liquid removal and rinsing liquid to remove to be combined in albumen and other impurity on the plain film of described glass fibre, described protein liquid removal contains the Tris-HCl that concentration is 5～50mmol/L, pH value 6.5～8.0,0.1 the dehydrated alcohol of～0.5mol/L guanidinium isothiocyanate and 5%～20%, described rinsing liquid contains the Tris-HCl that concentration is 5～50mmol/L, pH value 6.5～8.0,50～200mmol/LNaCl and 50%～80% dehydrated alcohol.Wherein, protein liquid removal can be removed albumen or other impurity of absorption on the plain film of described glass fibre or not absorption, and rinsing liquid can be removed impurity such as the albumen that adsorbs on the plain film of described glass fibre and other meta-bolitess such as polysaccharide, Mierocrystalline cellulose, lipid.
Wherein, as preferably, described protein liquid removal contains the Tris-HCl that concentration is 10mmol/L, pH value 7.5,0.3mol/L guanidinium isothiocyanate and 10% dehydrated alcohol.
As preferably, described rinsing liquid contains the Tris-HCl that concentration is 10mmol/L, pH value 7.5,100mmol/LNaCl and 80% dehydrated alcohol.
The method of extraction of the present invention RNA, the RNA on the plain film of the described glass fibre of step 4 wash-out can be with the eluant solution RNA of the high pH value of less salt of RNase-free.
Wherein, as preferably, with the pH value 0.1% the DEPC treating water of 7.0～8.5 sterilization or the sterile distilled water eluted rna of nuclease free.
More preferably, be 0.1% the DEPC treating water of 8.0 sterilization or the sterile distilled water eluted rna of nuclease free with the pH value.
The present invention also provides a kind of toxic reagents such as phenol, chloroform that do not contain, and can extract the test kit of high-quality RNA from multiple materials such as animal, plant and microorganism.
A kind of test kit that extracts RNA, comprise lysate, protein liquid removal, rinsing liquid, wherein, it is 10～50mmol/L that described lysate contains concentration, the Tris-HCl of pH value 6.5～8.0,3～5mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol, it is 5～50mmol/L that described protein liquid removal contains concentration, the Tris-HCl of pH value 6.5～8.0,0.1 the dehydrated alcohol of～0.5mol/L guanidinium isothiocyanate and 5%～20%, it is 5～50mmol/L that described rinsing liquid contains concentration, the Tris-HCl of pH value 6.5～8.0,50～200mmol/LNaCl and 50%～80% dehydrated alcohol.
The test kit of extraction of the present invention RNA, described lysate provides optimum buffer condition for the extraction of RNA, activity that can strongly inhibited RNase, efficient lysing cell release RNA again.Wherein, as preferably, described lysate contains the Tris-HCl that concentration is 25mmol/L, pH value 7.5,4mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol.
The test kit of extraction RNA of the present invention, described protein liquid removal can be removed albumen or other impurity that adsorbs or do not adsorb on the plain film of described glass fibre.Wherein, as preferably, described protein liquid removal contains the Tris-HCl that concentration is 10mmol/L, pH value 7.5,0.3mol/L guanidinium isothiocyanate and 10% dehydrated alcohol.
The test kit of extraction of the present invention RNA, described rinsing liquid can be removed impurity such as the albumen that adsorbs on the plain film of described glass fibre and other meta-bolitess such as polysaccharide, Mierocrystalline cellulose, lipid.Wherein, as preferably, described rinsing liquid contains the Tris-HCl that concentration is 10mmol/L, pH value 7.5,100mmol/LNaCl and 80% dehydrated alcohol.
The test kit of extraction RNA of the present invention also comprises the plain film of 0.45 μ m glass fibre, and the plain film of described 0.45 μ m glass fibre can selective adsorption RNA under the ethanol effect.The plain film of described 0.45 μ m glass fibre can be fixed in the centrifugal post, forms the plain film adsorption column of 0.45 μ m glass fibre.
The test kit of extraction RNA of the present invention also comprises elutriant, and described elutriant is the solution of the high pH value of the less salt of RNase-free, can elute the RNA that is adsorbed on the plain film of described glass fibre.
Wherein, as preferably, described elutriant is that the pH value is 0.1% the DEPC treating water of 7.0～8.5 sterilization or the sterile distilled water of nuclease free.
More preferably, described elutriant is that the pH value is 0.1% the DEPC treating water of 8.0 sterilization or the sterile distilled water eluted rna of nuclease free.
Many materials are rich in phenolic compound, polysaccharide and secondary metabolite that some still can't be determined, these materials are isolating with nucleic acid spatially in complete cell, but in extracting the RNA process, after tissue was by grinding, cytoclasis, these materials will interact with RNA.Can irreversibly combine with RNA after phenolic compound is oxidized, cause the RNA loss of activity; RNA easily loses with phenol, chloroform extracting the time, or forms insoluble mixture; And polysaccharide can form the jelly of indissoluble, gets off with the RNA co-precipitation; Terpenoid and RNase can cause chemical degradation and the enzymolysis of RNA respectively.Because polysaccharide polyphenol class material and RNA interact, and make that the separation and purification of RNA is extremely difficult, have hindered its molecular biology aspect progress of research.
Therefore another object of the present invention provides a kind of method that is applicable to the extraction RNA that is rich in polysaccharide polyphenol and secondary metabolite material.
A kind of method of extracting RNA comprises:
Step 1: sample and lysate mixing, centrifugal, add the extraction aid mixing in the supernatant, centrifugal, it is 10～50mmol/L that described lysate contains concentration, it is 5～20% polyvinylpyrrolidone and 0.5～2% Tween20 that the Tris-HCl of pH value 6.5～8.0,3～5mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol, described extraction aid contain concentration;
Step 2: get step 1 gained supernatant,, combine with the plain film of 0.45 μ m glass fibre with the dehydrated alcohol mixing of 1/2 volume RNase-free;
Step 3: wash the plain film of described glass fibre with protein liquid removal and rinsing liquid, it is 5～50mmol/L that described protein liquid removal contains concentration, the Tris-HCl of pH value 6.5～8.0,0.1～0.5mol/L guanidinium isothiocyanate and 5%～20% dehydrated alcohol, it is 5～50mmol/L that described rinsing liquid contains concentration, the Tris-HCl of pH value 6.5～8.0,50～200mmol/LNaCl and 50%～80% dehydrated alcohol;
Step 4: the RNA that adsorbs on the plain film of the described glass fibre of wash-out.
The present invention adds the extraction aid mixing in described sample and the centrifugal gained supernatant of lysate mixing, it is 5～20% polyvinylpyrrolidone and 0.5～2% Tween20 that described extraction aid contains concentration, polysaccharide polyphenol specificity combination in energy and the nucleic acid, can remove polysaccharide polyphenol class material to greatest extent after centrifugal, thereby from the material that is rich in polysaccharide polyphenol and secondary metabolite, be separated to high-quality RNA effectively.Wherein, as preferably, it is 20% polyvinylpyrrolidone and 1% Tween20 that described extraction aid contains concentration.
The present invention extracts RNA in Gramineae greenweed, aloe, tree peony, ruby flower, pine needle, the Chinese ilex material by the method that adopts classical TRIZOL method, guanidinium isothiocyanate/beta-mercaptoethanol cracking process and extraction RNA of the present invention respectively, and the method for finding the RNA of extraction of the present invention obviously is better than classical TRIZOL method and guanidinium isothiocyanate/beta-mercaptoethanol cracking process at the effect of the vegetable material extraction RNA that is rich in polysaccharide polyphenol.
The present invention also provides a kind of test kit that is applicable to the material extraction RNA that is rich in polysaccharide polyphenol and secondary metabolite.
A kind of test kit that extracts RNA, comprise lysate, extraction aid, protein liquid removal, rinsing liquid, wherein, it is 10～50mmol/L that described lysate contains concentration, the Tris-HCl of pH value 6.5～8.0,3～5mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol, it is 5～20% polyvinylpyrrolidone and 0.5～2% Tween20 that described extraction aid contains concentration, it is 5～50mmol/L that described protein liquid removal contains concentration, the Tris-HCl of pH value 6.5～8.0,0.1 the dehydrated alcohol of～0.5mol/L guanidinium isothiocyanate and 5%～20%, it is 5～50mmol/L that described rinsing liquid contains concentration, the Tris-HCl of pH value 6.5～8.0,50～200mmol/LNaCl and 50%～80% dehydrated alcohol.
The test kit of extraction RNA of the present invention, it is 5～20% polyvinylpyrrolidone and 0.5～2% Tween20 that described extraction aid contains concentration, can combine with the polysaccharide polyphenol specificity in the nucleic acid, can remove polysaccharide polyphenol class material to greatest extent after centrifugal, obtain high-quality RNA thereby from the material that is rich in polysaccharide polyphenol and secondary metabolite, separate effectively.Wherein, as preferably, it is 20% polyvinylpyrrolidone and 1% Tween20 that described extraction aid contains concentration.
The method simple and fast of extraction RNA of the present invention does not use toxic reagent, applied widely, except being applied to vegetable material RNA extraction, is equally applicable to the extraction of animal tissues, filamentous fungus, bacteria RNA.The method of extraction of the present invention RNA adds extraction aid and can separate from the material that is rich in polysaccharide polyphenol and secondary metabolite effectively and obtain high-quality RNA in lysing cell.The test kit of extraction RNA of the present invention does not contain poisonous reagent, applied widely, and effect is better than external RNA extraction test kit when extracting RNA at the vegetable material that is rich in polysaccharide polyphenol, and cost is low, is applicable to vast laboratory and scientific effort.
Description of drawings
Fig. 1 shows the method according to the embodiment of the invention 4 described extraction RNA, the 1% agarose gel electrophoresis figure of the RNA that extracts from plant, bacterium, yeast, animal material; The detected material of swimming lane 1-5 is respectively the RNA that extracts from Root and stem of Cholla, JM109 intestinal bacteria, yeast AH109 bacterial strain, mouse liver tissue, mouse kidney tissue.
Fig. 2 shows the method according to classical TRIZOL method, guanidinium isothiocyanate/β beta-mercaptoethanol cracking process (Qiagen method) and the embodiment of the invention 7 described extraction RNA, the 1% agarose gel electrophoresis figure of the RNA that extracts from Gramineae greenweed, aloe, tree peony, ruby flower, pine needle, Chinese ilex material;
The detected material of swimming lane 1,4,7,10,13,16 is respectively the RNA that extracts according to the TRIZOL method from greenweed, aloe, tree peony, ruby flower, pine needle, Chinese ilex vegetable material;
The detected material of swimming lane 2,5,8,11,14,17 is respectively the RNA that extracts according to guanidinium isothiocyanate/beta-mercaptoethanol cracking process (Qiagen method) from greenweed, aloe, tree peony, ruby flower, pine needle, Chinese ilex vegetable material;
The detected material of swimming lane 3,6,9,12,15,18 is respectively the RNA that extracts according to the method for the embodiment of the invention 7 described extraction RNA from greenweed, aloe, tree peony, ruby flower, pine needle, Chinese ilex vegetable material.
Fig. 3 shows the method according to the embodiment of the invention 7 described extraction RNA, the 1% agarose gel electrophoresis figure of the RNA that extracts from filamentous fungus.
Embodiment
The embodiment of the invention discloses method and the test kit of a kind of RNA of extraction.Those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Method of the present invention and product are described by preferred embodiment, the related personnel obviously can be in not breaking away from content of the present invention, spirit and scope to method as herein described with product is changed or suitably change and combination, realize and use the technology of the present invention.
For realizing purpose of the present invention, the present invention adopts following technical scheme:
Simple and fast of the present invention, do not use toxic reagent, can from multiple materials such as animal, plant and microorganism, extract the method for high-quality RNA, comprising:
Step 1: sample and lysate mixing, centrifugal, to remove uncracked cell residue.Described lysate contains the Tris-HCl that concentration is 10～50mmol/L, pH value 6.5～8.0,3～5mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol;
Step 2: draw the centrifugal back of step 1 gained supernatant,, join in the plain film adsorption column of 0.45 μ m glass fibre with the dehydrated alcohol mixing of 1/2 volume RNase-free, centrifugal, the abandoned stream fluid;
Step 3: add protein liquid removal in the adsorption column, centrifugal abandoned stream fluid, add rinsing liquid in the adsorption column then, centrifugal abandoned stream fluid, and then it is centrifugal to remove residual ethanol, described protein liquid removal contains the Tris-HCl that concentration is 5～50mmol/L, pH value 6.5～8.0,0.1 the dehydrated alcohol of～0.5mol/L guanidinium isothiocyanate and 5%～20%, described rinsing liquid contains the Tris-HCl that concentration is 5～50mmol/L, pH value 6.5～8.0,50～200mmol/LNaCl and 50%～80% dehydrated alcohol;
Step 4: add elutriant in adsorption column, room temperature is placed, and is centrifugal, collects effluent liquid and is the plant RNA of carrying, and described elutriant is the solution of the high pH value of the less salt of RNase-free.
The phenol that do not contain of the present invention, toxic reagents such as chloroform, can be from animal, extract the test kit of high-quality RNA in the multiple material such as plant and microorganism, comprise lysate, protein liquid removal, rinsing liquid, wherein, it is 10～50mmol/L that described lysate contains concentration, the Tris-HCl of pH value 6.5～8.0,3～5mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol, it is 5～50mmol/L that described protein liquid removal contains concentration, the Tris-HCl of pH value 6.5～8.0,0.1 the dehydrated alcohol of～0.5mol/L guanidinium isothiocyanate and 5%～20%, it is 5～50mmol/L that described rinsing liquid contains concentration, the Tris-HCl of pH value 6.5～8.0,50～200mmol/LNaCl and 50%～80% dehydrated alcohol.
Wherein, as preferably, described test kit also comprises plain film of 0.45 μ m glass fibre and elutriant, and described elutriant is the solution of the high pH value of the less salt of RNase-free.
The method that is applicable to the extraction RNA that is rich in polysaccharide polyphenol and secondary metabolite material of the present invention comprises:
Step 1: sample and lysate mixing, centrifugal, add the extraction aid mixing in the supernatant, centrifugal, it is 10～50mmol/L that described lysate contains concentration, it is 5～20% polyvinylpyrrolidone and 0.5～2% Tween20 that the Tris-HCl of pH value 6.5～8.0,3～5mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol, described extraction aid contain concentration;
Step 2: draw the centrifugal back of step 1 gained supernatant,, join in the plain film adsorption column of 0.45 μ m glass fibre with the dehydrated alcohol mixing of 1/2 volume RNase-free, centrifugal, the abandoned stream fluid;
Step 3: add protein liquid removal in the adsorption column, centrifugal abandoned stream fluid, add rinsing liquid in the adsorption column then, centrifugal abandoned stream fluid, and then it is centrifugal to remove residual ethanol, described protein liquid removal contains the Tris-HCl that concentration is 5～50mmol/L, pH value 6.5～8.0,0.1 the dehydrated alcohol of～0.5mol/L guanidinium isothiocyanate and 5%～20%, described rinsing liquid contains the Tris-HCl that concentration is 5～50mmol/L, pH value 6.5～8.0,50～200mmol/LNaCl and 50%～80% dehydrated alcohol;
Step 4: add elutriant in adsorption column, room temperature is placed, and is centrifugal, collects effluent liquid and is the plant RNA of carrying, and described elutriant is the solution of the high pH value of the less salt of RNase-free.
The test kit that is applicable to the material extraction RNA that is rich in polysaccharide polyphenol and secondary metabolite of the present invention, comprise lysate, extraction aid, protein liquid removal, rinsing liquid, wherein, it is 10～50mmol/L that described lysate contains concentration, the Tris-HCl of pH value 6.5～8.0,3～5mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol, it is 5～20% polyvinylpyrrolidone and 0.5～2% Tween20 that described extraction aid contains concentration, it is 5～50mmol/L that described protein liquid removal contains concentration, the Tris-HCl of pH value 6.5～8.0,0.1 the dehydrated alcohol of～0.5mol/L guanidinium isothiocyanate and 5%～20%, it is 5～50mmol/L that described rinsing liquid contains concentration, the Tris-HCl of pH value 6.5～8.0,50～200mmol/LNaCl and 50%～80% dehydrated alcohol.
Wherein, as preferably, described test kit also comprises plain film of 0.45 μ m glass fibre and elutriant, and described elutriant is the solution of the high pH value of the less salt of RNase-free.
The method simple and fast of extraction RNA of the present invention does not use toxic reagent, and is applied widely, except the extraction that is applied to vegetable material RNA, is equally applicable to the extraction of animal tissues, filamentous fungus, bacteria RNA.The method of extraction of the present invention RNA adds extraction aid and can separate from the material that is rich in polysaccharide polyphenol and secondary metabolite effectively and obtain high-quality RNA in lysing cell.The test kit of extraction RNA of the present invention does not contain poisonous reagent, applied widely, and effect is better than external RNA extraction test kit when extracting RNA at the vegetable material that is rich in polysaccharide polyphenol, and cost is low, is applicable to vast laboratory and scientific effort.
In order further to understand the present invention, method and the test kit that the present invention extracts RNA is elaborated below in conjunction with embodiment.
Embodiment 1: the test kit of extraction RNA of the present invention
The test kit of extraction RNA of the present invention comprises lysate, protein liquid removal, rinsing liquid.
Guanidinium isothiocyanate, 10mM pH value that lysate contains 3M are 6.5 Tris-HCl, 1% beta-mercaptoethanol.Concrete compound method is: take by weighing guanidinium isothiocyanate 35.45g, adds enough RNase-free distilled water and fully dissolve, add pH then and be 6.5, concentration is the Tris-HCl 1.0ml of 1M, abundant mixing, constant volume be to 99ml, adding 1ml beta-mercaptoethanol.
Guanidinium isothiocyanate, 5mM pH value that protein liquid removal contains 0.1M are 7.5 Tris-HCl, 5% dehydrated alcohol.Take by weighing the 1.18g guanidinium isothiocyanate, the distilled water that adds RNase-free fully dissolves, and adds pH then and be 6.5, concentration is the Tris-HCl 0.5ml of 1M, adds the dehydrated alcohol of 10ml RNase-free at last, and constant volume is to 100ml, abundant mixing.
The pH value that rinsing liquid contains 5mM is the NaCl of 6.5 Tris-HCl, 50mM, 50% dehydrated alcohol.Measuring the pH value is 6.5, and concentration is the Tris-HCl 0.5ml of 1M, measures the NaCl1ml of 5M again, adds the distilled water mixing of an amount of RNase-free, adds the dehydrated alcohol of the RNase-free of 50ml again, and constant volume is to 100ml, fully mixing.
Embodiment 2: the test kit of extraction RNA of the present invention
The test kit of extraction RNA of the present invention comprises lysate, protein liquid removal, rinsing liquid, the plain film of elutriant and glass fibre.Wherein, to contain guanidinium isothiocyanate, the 50mM pH value of 5M be 8.0 Tris-HCl, 1% beta-mercaptoethanol to lysate; Guanidinium isothiocyanate, 50mM pH value that protein liquid removal contains 0.5M are 8.0 Tris-HCl, 20% dehydrated alcohol; The pH value that rinsing liquid contains 20mM is the NaCl of 8.0 Tris-HCl, 200mM, 80% dehydrated alcohol; Elutriant is 0.1% a DEPC treating water of pH value 8.0 sterilization; The plain film of glass fibre is 0.45 μ m, Whatman company, GF/B type.Collocation method is with embodiment 1.
Embodiment 3: the test kit of extraction RNA of the present invention
The test kit of extraction RNA of the present invention comprises lysate, protein liquid removal, rinsing liquid, the plain film adsorption column of elutriant and glass fibre.Wherein, to contain guanidinium isothiocyanate, the 25mM pH value of 4M be 7.5 Tris-HCl, 1% beta-mercaptoethanol to lysate; Guanidinium isothiocyanate, 10mMpH value that protein liquid removal contains 0.3M are 7.5 Tris-HCl, 10% dehydrated alcohol; The pH value that rinsing liquid contains 10mM is the NaCl of 7.5 Tris-HCl, 100mM, 80% dehydrated alcohol; Elutriant is the sterile distilled water of pH value 7.5 nuclease free; The plain film adsorption column of glass fibre is the plain film of 0.45 μ m glass fibre, Whatman company, GF/B type, the centrifugal post of 1.5mL.Collocation method is with embodiment 1.
Embodiment 4: the method for extraction RNA of the present invention
Extract RNA with embodiment 3 described test kits.Sample adds lysate, abundant mixing, and room temperature is placed 5min and is made its abundant cracking, and then 12, the centrifugal 10min of 000rpm is to remove uncracked cell residue.Draw the supernatant after centrifugal, add the dehydrated alcohol of supernatant 1/2 volume RNase-free, fully mixing.Join in the plain film adsorption column of glass fibre, 12, the centrifugal 30s of 000rpm, abandoned stream fluid.Add protein liquid removal in the adsorption column, 12, the centrifugal 30s of 000rpm, abandoned stream fluid.Add rinsing liquid in the adsorption column then, 12, the centrifugal 30s of 000rpm, abandoned stream fluid.Add the rinsing liquid washing more once, the abandoned stream fluid, and then 12, the centrifugal 2min of 000rpm is to remove residual ethanol.Shift in the centrifuge tube of adsorption column to a clean RNase-free, add elutriant in adsorption column, room temperature is placed 1min, and 12, the centrifugal 1min of 000rpm collects effluent liquid.The effluent liquid of collecting joins in the adsorption column again and repeats wash-out once, collects effluent liquid, and effluent liquid is the plant RNA of carrying.
Embodiment 5: the test kit of extraction RNA of the present invention
The test kit of extraction RNA of the present invention comprises lysate, extraction aid, protein liquid removal, rinsing liquid, the plain film adsorption column of elutriant and glass fibre.Wherein, to contain guanidinium isothiocyanate, the 25mMpH value of 4M be 7.5 Tris-HCl, 1% beta-mercaptoethanol to lysate; Extraction aid contains 20% polyvinylpyrrolidone and 1% Tween20; Guanidinium isothiocyanate, 10mM pH value that protein liquid removal contains 0.3M are 7.5 Tris-HCl, 10% dehydrated alcohol; The pH value that rinsing liquid contains 10mM is the NaCl of 7.5 Tris-HCl, 100mM, 80% dehydrated alcohol; Elutriant is the sterile distilled water of pH value 7.5 nuclease free; The plain film adsorption column of glass fibre is the plain film of 0.45 μ m glass fibre, Whatman company, GF/B type, the centrifugal post of 1.5mL.The concrete compound method of extraction aid is: take by weighing 2 gram polyvinylpyrrolidones (PVP10), adding distil water fully dissolves, and draws the Tween20 of 100 μ l then with the most advanced and sophisticated suction nozzle of wiping out 0.5cm, abundant stirring and evenly mixing, and constant volume is to 10ml.Other reagent collocation methods are with embodiment 1.
Embodiment 6: the test kit of extraction RNA of the present invention
The test kit of extraction RNA of the present invention comprises lysate, extraction aid, protein liquid removal, rinsing liquid, the plain film of elutriant and glass fibre.Wherein, to contain guanidinium isothiocyanate, the 25mM pH value of 3M be 8.0 Tris-HCl, 1% beta-mercaptoethanol to lysate; Extraction aid contains 10% polyvinylpyrrolidone and 2% Tween20; Guanidinium isothiocyanate, 10mM pH value that protein liquid removal contains 0.3M are 8.0 Tris-HCl, 10% dehydrated alcohol; The pH value that rinsing liquid contains 10mM is the NaCl of 8.0 Tris-HCl, 100mM, 50% dehydrated alcohol; Elutriant is 0.1% a DEPC treating water of pH value 8.0 sterilization; The plain film of glass fibre is the plain film of 0.45 μ m glass fibre, Whatman company, GF/B type.Collocation method is with embodiment 4.
Embodiment 7: the method for extraction RNA of the present invention
Extract RNA with embodiment 5 described test kits.Sample adds lysate and extraction aid, abundant mixing, and room temperature is placed 5min and is made its abundant cracking, and then 12, the centrifugal 10min of 000rpm is to remove uncracked cell residue.Draw the supernatant after centrifugal, add the dehydrated alcohol of supernatant 1/2 volume RNase-free, fully mixing.Join in the plain film adsorption column of glass fibre, 12, the centrifugal 30s of 000rpm, abandoned stream fluid.Add protein liquid removal in the adsorption column, 12, the centrifugal 30s of 000rpm, abandoned stream fluid.Add rinsing liquid in the adsorption column then, 12, the centrifugal 30s of 000rpm, abandoned stream fluid.Add the rinsing liquid washing more once, the abandoned stream fluid, and then 12, the centrifugal 2min of 000rpm is to remove residual ethanol.Shift in the centrifuge tube of adsorption column to a clean RNase-free, add elutriant in adsorption column, room temperature is placed 1min, and 12, the centrifugal 1min of 000rpm collects effluent liquid.The effluent liquid of collecting joins in the adsorption column again and repeats wash-out once, collects effluent liquid, and effluent liquid is the plant RNA of carrying.
Embodiment 8: the method for extraction RNA of the present invention is extracted Root and stem of Cholla RNA
With reference to embodiment 4 described methods, extract Root and stem of Cholla RNA with embodiment 3 described test kits.Take by weighing the 0.1g Root and stem of Cholla, be cut into small pieces rapidly and put into mortar respectively, add the 1ml lysate, grind to form homogenate under the room temperature rapidly, room temperature is got supernatant behind the centrifugal 10min of room temperature after placing 5min, the dehydrated alcohol that in supernatant liquor, adds the RNase-free of 1/2 volume, fully mixing; Mixed solution is joined in the plain film adsorption column of glass fibre, 12, the centrifugal 30s of 000rpm, abandoned stream fluid; Add 500 μ l protein liquid removals in adsorption column, room temperature is placed 2min, 12, the centrifugal 30s of 000rpm, abandoned stream fluid; In adsorption column, add 500 μ l rinsing liquids, 12, the centrifugal 30s of 000rpm, the abandoned stream fluid repeats rinsing once, and again 12, the centrifugal 2min of 000rpm; Shift in the centrifuge tube of adsorption column to a clean RNase-free, add 100 μ l elutriants in the middle part of adsorption column, room temperature is placed 1min, 12, the centrifugal 1min of 000rpm, the effluent liquid of collecting joins in the adsorption column again and repeats wash-out once, collects effluent liquid, and effluent liquid is total RNA of the Root and stem of Cholla of carrying.1% agarose gel electrophoresis detects, and the result as shown in Figure 1.
Embodiment 9: the method for extraction RNA of the present invention is extracted bacteria RNA
With reference to embodiment 4 described methods, extract bacteria RNA with embodiment 3 described test kits.Centrifugal collection 1-2ml Escherichia coli bacteria liquid (10
8～10
9Cell) to a 1.5ml centrifuge tube, remove supernatant as far as possible, in cell precipitation, add the TE that 100 μ l contain the 1mg/ml N,O-Diacetylmuramidase, 37 ℃ of incubation 15min, during every the vibration of 5min vortex once; Add 350 μ l lysates, abundant vortex mixing, 12, the centrifugal 3min of 000rpm shifts supernatant to a new centrifuge tube, adds the dehydrated alcohol of supernatant 1/2 volume RNase-free again, fully mixing; Mixed solution is joined in the plain film adsorption column of glass fibre, 12, the centrifugal 30s of 000rpm, abandoned stream fluid; Add 500 μ l protein liquid removals in adsorption column, room temperature is placed 2min, 12, the centrifugal 30s of 000rpm, abandoned stream fluid; In adsorption column, add 500 μ l rinsing liquids, 12, the centrifugal 30s of 000rpm, the abandoned stream fluid repeats rinsing once, and again 12, the centrifugal 2min of 000rpm; Shift in the centrifuge tube of adsorption column to a clean RNase-free, add 100 μ l elutriants in the middle part of adsorption column, room temperature is placed 1min, 12, the centrifugal 1min of 000rpm, the effluent liquid of collecting joins in the adsorption column again and repeats wash-out once, collects effluent liquid, and effluent liquid is total RNA of the bacterium of carrying.1% agarose gel electrophoresis detects, and the result as shown in Figure 1.
Embodiment 10: the method for extraction RNA of the present invention is extracted yeast rna
With reference to embodiment 4 described methods, extract yeast rna with embodiment 3 described test kits.Collect 1ml (about 10
7Cell) yeast culture that is in logarithmic phase is in the 1.5ml centrifuge tube, and 9, the centrifugal 30s of 000rpm removes supernatant as far as possible, adds 100 μ l damping fluid SE (1M sorbyl alcohol, 0.1M Na
2EDTA, the 14mM beta-mercaptoethanol), softly blow and beat abundant re-suspended cell; Add about 50U lyticase according to the yeast amount, fully put upside down mixing, 37 ℃ of incubation 10-30min peptic cell walls, the centre can be put upside down and be helped digest for several times, adds the violent vortex piping and druming of 350 μ l lysates mixing then, 12, the centrifugal 3min of 000rpm, shift supernatant to a new centrifuge tube, add the dehydrated alcohol of supernatant 1/2 volume RNase-free, blow and beat mixing immediately; Mixed solution is joined in the plain film adsorption column of glass fibre, 12, the centrifugal 30s of 000rpm, abandoned stream fluid; Add 500 μ l protein liquid removals in adsorption column, room temperature is placed 2min, 12, the centrifugal 30s of 000rpm, abandoned stream fluid; In adsorption column, add 500 μ l rinsing liquids, 12, the centrifugal 30s of 000rpm, the abandoned stream fluid repeats rinsing once, and again 12, the centrifugal 2min of 000rpm; Shift in the centrifuge tube of adsorption column to a clean RNase-free, add 100 μ l elutriants in the middle part of adsorption column, room temperature is placed 1min, 12, the centrifugal 1min of 000rpm, the effluent liquid of collecting joins in the adsorption column again and repeats wash-out once, collects effluent liquid, and effluent liquid is the total RNA of institute's zymic of carrying.1% agarose gel electrophoresis detects, and the result as shown in Figure 1.
Embodiment 11: the method for extraction RNA of the present invention is extracted mouse liver and kidney RNA
With reference to embodiment 4 described methods, extract mouse liver and kidney RNA with embodiment 3 described test kits.Take by weighing mouse kidney or liver organization 0.1g, in liquid nitrogen, grind to form fine powder rapidly, ground fine powder is transferred in the 1.5ml centrifuge tube that 350 μ l lysates are housed, and vortex vibrates into homogenate, with homogenate 12, the centrifugal 3min of 000rpm, shift supernatant to a new centrifuge tube, add the dehydrated alcohol of supernatant 1/2 volume RNase-free, precipitation may appear in this moment, but do not influence leaching process, blow and beat mixing immediately; Mixed solution is joined in the plain film adsorption column of glass fibre, 12, the centrifugal 30s of 000rpm, abandoned stream fluid; Add 500 μ l protein liquid removals in adsorption column, room temperature is placed 2min, 12, the centrifugal 30s of 000rpm, abandoned stream fluid; In adsorption column, add 500 μ l rinsing liquids, 12, the centrifugal 30s of 000rpm, the abandoned stream fluid repeats rinsing once, and again 12, the centrifugal 2min of 000rpm; Shift in the centrifuge tube of adsorption column to a clean RNase-free, add 100 μ l elutriants in the middle part of adsorption column, room temperature is placed 1min, 12, the centrifugal 1min of 000rpm, the effluent liquid of collecting joins in the adsorption column again and repeats wash-out once, collects effluent liquid, and effluent liquid is total RNA of the renal tissue of carrying.1% agarose gel electrophoresis detects, and the result as shown in Figure 1.
Embodiment 12: the method for extraction RNA of the present invention is extracted the material RNA that is rich in the polysaccharide polyphenol secondary metabolite
With reference to embodiment 7 described methods, extract the material RNA that is rich in the polysaccharide polyphenol secondary metabolite with embodiment 5 described test kits.Take by weighing each three parts of plants such as the fresh greenweed of 0.1g, aloe, tree peony, ruby flower, pine needle, Chinese ilex, be cut into small pieces rapidly and put into mortar respectively, first part is extracted RNA with the described methods of embodiment 7, add 1ml lysate and 100 μ l extraction aids, grind to form homogenate under the room temperature rapidly, second part of classical Trizol reagent of adding 1ml directly grinds to form homogenate, and the 3rd part of lysate that adds 1ml guanidinium isothiocyanate/beta-mercaptoethanol cracking process directly grinds to form homogenate.After room temperature was placed 5min, classical TRIZOL method and guanidinium isothiocyanate/beta-mercaptoethanol cracking process carried out the extraction of RNA according to the operation steps of routine.The 3rd part is extracted RNA with the described methods of embodiment 7, adds the dehydrated alcohol of the RNase-free of 1/2 times of volume in supernatant liquor, fully mixing; Mixed solution is joined in the plain film adsorption column of glass fibre, 12, the centrifugal 30s of 000rpm, abandoned stream fluid; Add 500 μ l protein liquid removals in adsorption column, room temperature is placed 2min, 12, the centrifugal 30s of 000rpm, abandoned stream fluid; In adsorption column, add 500 μ l rinsing liquids, 12, the centrifugal 30s of 000rpm, the abandoned stream fluid repeats rinsing once, and 12, the centrifugal 2min of 000rpm; Shift in the centrifuge tube of adsorption column to a clean RNase-free, add 100 μ l elutriants in the middle part of adsorption column, room temperature is placed 1min, 12, the centrifugal 1min of 000rpm, the effluent liquid of collecting joins in the adsorption column again and repeats wash-out once, collects effluent liquid, and effluent liquid is total RNA of the various materials of carrying.1% agarose gel electrophoresis detects, and the result as shown in Figure 2.
Embodiment 13: the method for extraction RNA of the present invention is extracted filamentous fungus RNA
With reference to embodiment 7 described methods, extract filamentous fungus RNA with embodiment 5 described test kits.Take by weighing 0.1g filamentous fungus mycelia, in liquid nitrogen, grind to form fine powder rapidly, ground fine powder is transferred in the 1.5ml centrifuge tube that 500 μ l lysates are housed, add 50 μ l extraction aids, vortex vibrates into homogenate, with homogenate 12, the centrifugal 3min of 000rpm, shift supernatant to a new centrifuge tube, add the dehydrated alcohol of the RNase-free of 0.5 times of volume of supernatant, blow and beat mixing immediately; Mixed solution is joined in the plain film adsorption column of glass fibre, 12, the centrifugal 30s of 000rpm, abandoned stream fluid; Add 500 μ l protein liquid removals in adsorption column, room temperature is placed 2min, 12, the centrifugal 30s of 000rpm, abandoned stream fluid; In adsorption column, add 500 μ l rinsing liquids, 12, the centrifugal 30s of 000rpm, the abandoned stream fluid repeats rinsing once, and again 12, the centrifugal 2min of 000rpm; Shift in the centrifuge tube of adsorption column to a clean RNase-free, add 100 μ l elutriants in the middle part of adsorption column, room temperature is placed 1min, 12, the centrifugal 1min of 000rpm, the effluent liquid of collecting joins in the adsorption column again and repeats wash-out once, collects effluent liquid, and effluent liquid is total RNA of the filamentous fungus mycelia of carrying.1% agarose gel electrophoresis detects, and the result as shown in Figure 3.
The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Claims (10)
1. method of extracting RNA comprises:
Step 1: sample and lysate mixing, centrifugal, it is 10～50mmol/L that described lysate contains concentration, the Tris-HCl of pH value 6.5～8.0,3～5mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol;
Step 2: get step 1 gained supernatant,, combine with the plain film of 0.45 μ m glass fibre with the dehydrated alcohol mixing of 1/2 volume RNase-free;
Step 3: wash the plain film of described glass fibre with protein liquid removal and rinsing liquid, it is 5～50mmol/L that described protein liquid removal contains concentration, the Tris-HCl of pH value 6.5～8.0,0.1～0.5mol/L guanidinium isothiocyanate and 5%～20% dehydrated alcohol, it is 5～50mmol/L that described rinsing liquid contains concentration, the Tris-HCl of pH value 6.5～8.0,50～200mmol/LNaCl and 50%～80% dehydrated alcohol;
Step 4: the RNA that adsorbs on the plain film of the described glass fibre of wash-out.
2. according to the described method of claim 1, it is characterized in that it is 25mmol/L that described lysate contains concentration, the Tris-HCl of pH value 7.5,4mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol.
3. according to claim 1 or 2 described methods, it is characterized in that step 1 also comprises gained supernatant and extraction aid mixing, and is centrifugal, it is 5～20% polyvinylpyrrolidone and 0.5～2% Tween20 that described extraction aid contains concentration.
4. method according to claim 3 is characterized in that, it is 20% polyvinylpyrrolidone and 1% Tween20 that described extraction aid contains concentration.
5. test kit that extracts RNA, comprise lysate, protein liquid removal, rinsing liquid, wherein, it is 10～50mmol/L that described lysate contains concentration, the Tris-HCl of pH value 6.5～8.0,3～5mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol, it is 5～50mmol/L that described protein liquid removal contains concentration, the Tris-HCl of pH value 6.5～8.0,0.1 the dehydrated alcohol of～0.5mol/L guanidinium isothiocyanate and 5%～20%, it is 5～50mmol/L that described rinsing liquid contains concentration, the Tris-HCl of pH value 6.5～8.0,50～200mmol/LNaCl and 50%～80% dehydrated alcohol.
6. according to the described test kit of claim 5, it is characterized in that, also comprise the plain film of 0.45 μ m glass fibre.
7. according to claim 5 or 6 described test kits, it is characterized in that it is 25mmol/L that described lysate contains concentration, the Tris-HCl of pH value 7.5,4mol/L guanidinium isothiocyanate and 1% beta-mercaptoethanol.
8. according to any described test kit of claim 5-7, it is characterized in that, also comprise extraction aid, it is 5～20% polyvinylpyrrolidone and 0.5～2% Tween20 that described extraction aid contains concentration.
9. described according to Claim 8 test kit is characterized in that, it is 20% polyvinylpyrrolidone and 1% Tween20 that described extraction aid contains concentration.
10. according to any described test kit of claim 5-9, it is characterized in that also comprise elutriant, described elutriant is the solution of the high pH value of the less salt of RNase-free.
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| CN102634508A (en) * | 2012-04-24 | 2012-08-15 | 西北农林科技大学 | Method for extracting total RNA (Ribonucleic Acid) of grapholita molesta adult antennae |
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| CN103421764A (en) * | 2013-03-07 | 2013-12-04 | 华中农业大学 | Kit for quickly extracting double-stranded RNA of mycovirus and application of kit |
| CN103421764B (en) * | 2013-03-07 | 2015-06-03 | 华中农业大学 | Kit for quickly extracting double-stranded RNA of mycovirus and application of kit |
| CN103981173A (en) * | 2014-04-10 | 2014-08-13 | 盐城师范学院 | Extraction method for total RNA from secondary follicle tissue of Cashmere goat |
| CN106164270A (en) * | 2014-04-11 | 2016-11-23 | 和光纯药工业株式会社 | Nucleic acid purification method |
| CN105420227A (en) * | 2015-12-28 | 2016-03-23 | 中国科学院南海海洋研究所 | Hermatypic coral total RNA extraction method |
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