CN102329792B - Method for quickly extracting total ribonucleic acid (RNA) from Rubus - Google Patents
Method for quickly extracting total ribonucleic acid (RNA) from Rubus Download PDFInfo
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Abstract
The invention discloses a method for quickly extracting total ribonucleic acid (RNA) from Rubus. The method is characterized by comprising the following steps of: grinding a small amount of Rubus material into powder under liquid nitrogen freezing conditions, and optionally removing sugar and centrifuging, or directly adding an alkaline cell lysis solution and lysing in warm bath; performing phenol-weak acid-chloroform extraction after the lysis is finished, and centrifuging to remove proteins and most of genomic deoxyribonucleic acid (DNA); adding alcohol into supernatant and precipitating; and centrifuging and collecting a RNA precipitate, washing by using ethanol, air-drying, dissolving by using diethylprocarbonate (DEPC) sterile water, and preserving at the temperature of -80 DEG C. The use of a LiCl precipitation method is avoided, the proteins and the genomic DNA are removed through one-step extraction, the RNA is precipitated by using the alcohol, the method is easy to operate, and the whole process is finished within 3 hours; and only 0.1 to 0.2g of material is required when the total RNA is extracted from the Rubus by the method, the optional step of removing the sugar is added, and the method is applicable to various plant materials of the Rubus such as raspberries, blackberries and the like.
Description
(1) technical field
The invention belongs to the molecular biology of plants field, be specifically related to the method for the total RNA of a kind of rapid extraction rubus
(2) background technology
Obtaining the total RNA of high quality is the precondition of carrying out molecular biology research such as gene expression dose research, genetic manipulation such as the downstream researchs such as RNA interference, transgenosis.The polyphenol secondary metabolic products such as Kuromanine, pycnogenols have been rich in the raspberry of rubus, the blackberry, these materials extract for total RNA and brought numerous difficulties: phenolic compound very easily is oxidized to brown material, then irreversibly be combined with RNA, cause the RNA loss of activity, or form insoluble mixture and cause losing in a large number of with chloroform extracting time RNA.
The commercial reagents box, when extracting, the total RNA of such plant of reply seems helpless such as the Trizol test kit, and traditional method for extracting total RNA such as CTAB, SDS etc. need more vegetable material, and need to use the multistep extracting to remove polysaccharide, albumen and genomic dna.Using maximum methods at present is after the vegetable cell fragmentation, uses LiCl to carry out the selective precipitation of RNA, reaches the purpose of removing genomic dna or polysaccharide.But the shortcoming of the method maximum is consuming time oversize, and generally all selective precipitation is spent the night.If make the alcohol precipitation into, DNA pollutes especially severe, needs further to use DNase to process, and then passes through the phenol-chloroform extracting with deactivation DNase, and the result causes a large amount of losses of RNA.In addition, the elution process after the LiCl precipitation is strict, because residual Li
+, Cl
-Can produce negative impact to follow-up reverse transcription and pcr amplification.
So we are in the urgent need to a kind of more convenient and quicker, and the effective RNA extracting method of the materials such as Polysaccharide removing, polyphenol and genomic dna.
(3) summary of the invention
The invention a kind of be applicable to rubus simply, method for extracting total RNA fast.The method is characterized in that according to the following steps and carry out:
1) gets centrifuge tube, in centrifuge tube, add 700～1000 μ l desaccharification damping fluids, and add 5～15 μ l beta-mercaptoethanols, in-20 ℃ of precooling 5～10min;
2) get the vegetable material 0.1～0.15g of fresh plant material or-80 ℃ of preservations behind liquid nitrogen flash freezer, add the 0.1g cross-linking polyethylene pyrrolidone, under the liquid nitrogen freezing condition, grind to form powdery, be transferred to rapidly step 1) centrifuge tube in, tenderness turns upside down behind the mixing and to leave standstill 10min on ice;
3) under 4 ℃ according to the centrifugal 5～10min of 3200 * g;
4) thoroughly remove step 3) centrifugal gained supernatant liquor, in precipitation, add the cell pyrolysis liquid of 65 ℃ of preheatings of 700～1000 μ l, vortex 1min is in 55～65 ℃ of insulation 20～30min;
5) until step 4) in mixed solution be cooled to room temperature after, be sequentially added into the isopyknic water-saturated phenol of cell pyrolysis liquid, the pH adjusting agent of 1/80-1/50 cell pyrolysis liquid volume, the chloroform of 1/5 cell pyrolysis liquid volume, concuss 2min;
6) under 4 ℃ according to the centrifugal 5～10min of 16000 * g;
7) with step 6) centrifugal gained supernatant liquor changes another centrifuge tube over to, and Xiang Guanzhong adds an amount of nucleic acid precipitation agent, leaves standstill more than the 30min in-20 ℃, is abandoning supernatant liquor according to 16000 * g behind the centrifugal 10min under 4 ℃, keeps precipitation;
8) with step 7) centrifugal gained precipitation is that room temperature is air-dry after 70～75% washing with alcohol with volume fraction, with-80 ℃ of preservations after the DEPC processing water dissolution;
Wherein, step 1), 2), 3), 4) can be replaced by step:
9) get centrifuge tube, in centrifuge tube, add 700～1000 μ l cell pyrolysis liquids, and add 5～15 μ l beta-mercaptoethanols, in 55～65 ℃ of preheating 5～10min;
10) get the vegetable material 0.1～0.15g of-80 ℃ of preservations behind fresh plant material or the liquid nitrogen flash freezer, add the 0.1g cross-linking polyethylene pyrrolidone, under the liquid nitrogen freezing condition, grind to form powdery, be transferred to rapidly step 9) centrifuge tube in, vortex 1min is in 55～65 ℃ of insulation 20～30min;
Press such scheme, described desaccharification damping fluid is: the disodium ethylene diamine tetraacetate of 0.05M sodium-chlor, 50mM pH 8.0, the Tutofusin tris of 200mM pH 8.0.Cell pyrolysis liquid is: the disodium ethylene diamine tetraacetate of 30g/L cetyl trimethylammonium bromide, 1.4M sodium-chlor, 50～100mM pH 8.0, the Tutofusin tris of 100～200mM pH 8.0 and 20g/L PVP K30.PH adjusting agent includes but not limited to weak acid such as Glacial acetic acid, oxalic acid.Described nucleic acid precipitation agent includes but not limited to dehydrated alcohol, Virahol, and the dehydrated alcohol volume that uses is 2～2.5 times of the supernatant volume, uses the Virahol volume to be 0.8～1 times of the supernatant volume.
Press such scheme, described method is applicable to raspberry, blackberry, blueberry and other kinds or the mutation under the rubus.Described vegetable material comprises blade, flower, bud and fruit.
Usefulness of the present invention: compare with existing CTAB method, the present invention can the total RNA of each organ-tissue of rapid extraction rubus, and the time spent is short, can easily finish within 3 hours, and existing method needs more than 12 hours.For reaching the purpose of removing protein, polysaccharide and genomic dna, traditional method needs to take turns extracting and 2 through 2～3 and takes turns above precipitation, and complex operation step, present method are only carried out 1 and taken turns extracting and 1 and take turns precipitation, have simplified operating process.Present method increased optional desaccharification step of a step before Deproteinization, can extract in the material sugar content according to RNA and just select, and had increased the suitability of method, can be applicable to the plants such as rubus raspberry, blackberry, blueberry.
(4) description of drawings
Fig. 1 is the total RNA gel electrophoresis figure of blackberry, blueberry fruit that adopts present method to extract;
L is the standard molecular weight reference among Fig. 1, and 1～4 is respectively 4 repetitions adopting the total RNA of blackberry, blueberry fruit of present method extraction, and 4000,2200 and 1000 point to respectively the pillar location of 4000,2200 and 1000 Nucleotide in the standard molecular weight reference;
Fig. 2 is after adopting the total RNA reverse transcription of blackberry, blueberry fruit of present method extraction, the pcr amplification gel electrophoresis figure of blackberry, blueberry chalcone synthase genes CHS and Actin muscle β-actin gene;
M2 and M1 are the standard molecular weight references among Fig. 2, and 1 and 2 is respectively to adopt the total RNA of blackberry, blueberry fruit that present method extracts after reverse transcription, the pcr amplification gel electrophoresis position of blackberry, blueberry chalcone synthase genes CHS; The 3rd, adopt the total RNA of blackberry, blueberry fruit of present method extraction after reverse transcription, the pcr amplification gel electrophoresis position of blackberry, blueberry Actin muscle β-actin gene; 100,300,500,700,900 and 1200 point to respectively standard molecular weight with reference to 100,300 among the M2, the pillar location of 500,700,900 and 1200 Nucleotide; 100,200,300,400,500 and 600 point to respectively standard molecular weight with reference to 100,200 among the M1, the pillar location of 300,400,500 and 600 Nucleotide.
(5) embodiment
1, the present invention implements according to the following steps:
1) gets centrifuge tube, in centrifuge tube, add 700～1000 μ l desaccharification damping fluids, and add 5～15 μ l beta-mercaptoethanols, in-20 ℃ of precooling 5～10min;
2) get the vegetable material 0.1～0.15g of-80 ℃ of preservations behind fresh plant material or the liquid nitrogen flash freezer, add the 0.1g cross-linking polyethylene pyrrolidone, under the liquid nitrogen freezing condition, grind to form powdery, be transferred to rapidly step 1) centrifuge tube in, tenderness turns upside down behind the mixing and to leave standstill 10min on ice;
3) under 4 ℃ according to the centrifugal 5～10min of 3200 * g;
4) thoroughly remove step 3) centrifugal gained supernatant liquor, in precipitation, add the cell pyrolysis liquid of 65 ℃ of preheatings of 700～1000 μ l, vortex 1min is in 55～65 ℃ of insulation 20～30min;
5) until step 4) in mixed solution be cooled to room temperature after, be sequentially added into the isopyknic water-saturated phenol of cell pyrolysis liquid, the pH adjusting agent of 1/80-1/50 cell pyrolysis liquid volume, the chloroform of 1/5 cell pyrolysis liquid volume, concuss 2min;
6) under 4 ℃ according to the centrifugal 5～10min of 16000 * g;
7) with step 6) centrifugal gained supernatant liquor changes another centrifuge tube over to, and Xiang Guanzhong adds an amount of nucleic acid precipitation agent, leaves standstill more than the 30min in-20 ℃, is abandoning supernatant liquor according to 16000 * g behind the centrifugal 10min under 4 ℃, keeps precipitation;
8) with step 7) centrifugal gained precipitation is that room temperature is air-dry after 70～75% washing with alcohol with volume fraction, with-80 ℃ of preservations after the DEPC processing water dissolution;
Wherein, step 1), 2), 3), 4) can be replaced by step:
9) get centrifuge tube, in centrifuge tube, add 700～1000 μ l cell pyrolysis liquids, and add 5～15 μ l beta-mercaptoethanols, in 55～65 ℃ of preheating 5～10min;
10) get the vegetable material 0.1～0.15g of-80 ℃ of preservations behind fresh plant material or the liquid nitrogen flash freezer, add the 0.1g cross-linking polyethylene pyrrolidone, under the liquid nitrogen freezing condition, grind to form powdery, be transferred to rapidly step 9) centrifuge tube in, vortex 1min, 55～65 ℃ of insulation 20～30min;
Press such scheme, described desaccharification damping fluid is: the disodium ethylene diamine tetraacetate of 0.05M sodium-chlor, 50mM pH 8.0, the Tutofusin tris of 200mM pH 8.0.Cell pyrolysis liquid is: the disodium ethylene diamine tetraacetate of 30g/L cetyl trimethylammonium bromide, 1.4M sodium-chlor, 50～100mM pH 8.0, the Tutofusin tris of 100～200mM pH 8.0 and 20g/L PVP K30.PH adjusting agent includes but not limited to weak acid such as Glacial acetic acid, oxalic acid.Described nucleic acid precipitation agent includes but not limited to dehydrated alcohol, Virahol, and the dehydrated alcohol volume that uses is 2～2.5 times of the supernatant volume, uses the Virahol volume to be 0.8～1 times of the supernatant volume.
2, embodiment 1:
1) gets the 2ml centrifuge tube, in centrifuge tube, add 1000 μ l desaccharification damping fluids, and add 5 μ l beta-mercaptoethanols, in-20 ℃ of precooling 10min; 2) get the blackberry, blueberry fruit 0.1～0.15g of-80 ℃ of preservations behind the liquid nitrogen flash freezer, add the 0.1g cross-linking polyethylene pyrrolidone, under the liquid nitrogen freezing condition, grind to form powdery, be transferred to rapidly step 1) centrifuge tube in, tenderness turns upside down behind the mixing and to leave standstill 10min on ice; 3) under 4 ℃ according to the centrifugal 10min of 3200 * g; 4) thoroughly remove step 3) centrifugal gained supernatant liquor, add the cell pyrolysis liquid of 65 ℃ of preheatings of 700 μ l in the precipitation, vortex 1min is in 60 ℃ of insulation 30min; 5) until step 4) in mixed solution be cooled to room temperature after, be sequentially added into water-saturated phenol 700 μ l, 13 μ l Glacial acetic acid, 140 μ l chloroforms, concuss 2min; 6) under 4 ℃ according to the centrifugal 10min of 16000 * g; 7) with step 6) centrifugal gained supernatant liquor 500 μ l change another centrifuge tube over to, Xiang Guanzhong adds the Virahol of 500 μ l-20 ℃ precoolings, turn upside down behind the mixing in-20 ℃ and leave standstill 30min, abandoning supernatant liquor according to 16000 * g behind the centrifugal 10min under 4 ℃, keep precipitation; 8) with step 7) centrifugal gained precipitation is that room temperature is air-dry after 75% washing with alcohol with volume fraction, processes afterwards-80 ℃ of preservations of water 30 μ l dissolving with DEPC;
Press such scheme, described desaccharification damping fluid is: the disodium ethylene diamine tetraacetate of 0.05M sodium-chlor, 50mM pH 8.0, the Tutofusin tris of 200mM pH 8.0.Cell pyrolysis liquid is: the disodium ethylene diamine tetraacetate of 30g/L cetyl trimethylammonium bromide, 1.4M sodium-chlor, 50～100mM pH 8.0, the Tutofusin tris of 100～200mM pH 8.0 and 20g/L PVP K30.
3, embodiment 2:
1) gets the 2ml centrifuge tube, in centrifuge tube, add 700 μ l lysis buffers, and add 5 μ l beta-mercaptoethanols, in 60 ℃ of preheating 10min; 2) get fresh Leaves From Raspberry 0.1～0.15g, add the 0.1g cross-linking polyethylene pyrrolidone, under the liquid nitrogen freezing condition, grind to form powdery, be transferred to rapidly step 1) centrifuge tube in, vortex 1min is in 60 ℃ of insulation 30min; 3) until step 2) in mixed solution be cooled to room temperature after, be sequentially added into water-saturated phenol 700 μ l, 12 μ l Glacial acetic acid, 140 μ l chloroforms, concuss 2min; 4) under 4 ℃ according to the centrifugal 10min of 16000 * g; 5) with step 4) centrifugal gained supernatant liquor 500 μ l change another centrifuge tube over to, Xiang Guanzhong adds the dehydrated alcohol of 1250 μ l-20 ℃ precoolings, turn upside down behind the mixing in-20 ℃ and leave standstill 30min, abandoning supernatant liquor according to 16000 * g behind the centrifugal 10min under 4 ℃, keep precipitation; 6) with step 5) centrifugal gained precipitation is that room temperature is air-dry after 75% washing with alcohol with volume fraction, processes afterwards-80 ℃ of preservations of water 50 μ l dissolving with DEPC;
Press such scheme, described lysis buffer is: the disodium ethylene diamine tetraacetate of 30g/L cetyl trimethylammonium bromide, 1.4M sodium-chlor, 50～100 mM pH 8.0, the Tutofusin tris of 100～200mM pH 8.0 and 20g/L PVP K30.
4, implementation result
What Fig. 1 showed is the complete RNA band of the total RNA of blackberry, blueberry fruit that adopts present method to extract, and band is clear, bright, and without degraded, integrity is good, and the genomic dna band is not obvious, illustrates that RNA purity is high.
Fig. 2 is that the cDNA of the total RNA reverse transcription of blackberry, blueberry fruit of employing present method extraction is template, the blackberry, blueberry chalcone synthase genes CHS of pcr amplification and Actin muscle β-actin gene, band is clear bright, illustrates that this RNA can be applicable to the requirement of experiment such as clone, expression thereafter fully.
Table 1 pollutes without albumen, polysaccharide for yield and the purity of blackberry, blueberry blade, flower and the total RNA of fruit of present method extraction, the visible total RNA that extracts, and quality meets follow-up service requirements.
Yield and the purity of table 1 blackberry, blueberry blade, flower and the total RNA of fruit
Claims (6)
1. the method for the total RNA of rapid extraction rubus is characterized in that following operation:
(1) gets the 2ml centrifuge tube, in centrifuge tube, add 700～1000 μ l desaccharification damping fluids, and add 5～15 μ l beta-mercaptoethanols, in-20 ℃ of precooling 5～10min; Described desaccharification damping fluid is: the disodium ethylene diamine tetraacetate of 0.05M sodium-chlor, 50mM pH 8.0, the Tutofusin tris of 200mM pH 8.0;
(2) get the vegetable material 0.1～0.15g of fresh plant material or-80 ℃ of preservations behind liquid nitrogen flash freezer, add the 0.1g cross-linking polyethylene pyrrolidone, under the liquid nitrogen freezing condition, grind to form powdery, be transferred to rapidly in the centrifuge tube of step (1), tenderness turns upside down behind the mixing and to leave standstill 10min on ice;
(3) under 4 ℃ according to the centrifugal 5～10min of 3200 * g;
(4) thoroughly remove the centrifugal gained supernatant liquor of step (3), add the cell pyrolysis liquid of 65 ℃ of preheatings of 700～1000 μ l in precipitation, vortex 1min is in 55～65 ℃ of insulation 20～30min; Described cell pyrolysis liquid is: the disodium ethylene diamine tetraacetate of 30g/L cetyl trimethylammonium bromide, 1.4M sodium-chlor, 50～100mMpH 8.0, the Tutofusin tris of 100～200mMpH 8.0 and 20g/L PVP K30;
(5) after the mixed solution in step (4) is cooled to room temperature, be sequentially added into the isopyknic water-saturated phenol of cell pyrolysis liquid, the pH adjusting agent of 1/80-1/50 cell pyrolysis liquid volume, the chloroform of 1/5 cell pyrolysis liquid volume, concuss 2min; Described pH adjusting agent is weak acid;
(6) under 4 ℃ according to the centrifugal 5～10min of 16000 * g;
(7) change the centrifugal gained supernatant liquor of step (6) over to another centrifuge tube, Xiang Guanzhong adds an amount of nucleic acid precipitation agent, leaves standstill more than the 30min in-20 ℃, is abandoning supernatant liquor according to 16000 * g behind the centrifugal 10min under 4 ℃, keeps precipitation; Described nucleic acid precipitation agent is dehydrated alcohol or Virahol;
(8) be that room temperature is air-dry after 70～75% washing with alcohol with the centrifugal gained of step (7) precipitation with volume fraction, with-80 ℃ of preservations after the DEPC processing water dissolution.
2. the method for the total RNA of rapid extraction rubus according to claim 1, described step (1), (2), (3), (4) are replaced by:
(1) gets the 2ml centrifuge tube, in centrifuge tube, add 700～1000 μ l cell pyrolysis liquids, and add 5～15 μ l beta-mercaptoethanols, in 55～65 ℃ of preheating 5～10min; Described cell pyrolysis liquid is: the disodium ethylene diamine tetraacetate of 30g/L cetyl trimethylammonium bromide, 1.4M sodium-chlor, 50～100mM pH 8.0, the Tutofusin tris of 100～200mM pH 8.0 and 20g/L PVP K30;
(2) get the vegetable material 0.1～0.15g of-80 ℃ of preservations behind fresh plant material or the liquid nitrogen flash freezer, add the 0.1g cross-linking polyethylene pyrrolidone, under the liquid nitrogen freezing condition, grind to form powdery, be transferred to rapidly in the centrifuge tube of step (1), vortex 1min is in 55～65 ℃ of insulation 20～30min.
3. the method for the total RNA of rapid extraction rubus according to claim 1 and 2, it is characterized in that: pH adjusting agent is Glacial acetic acid or oxalic acid described in the step (5).
4. the method for the total RNA of rapid extraction rubus according to claim 1 and 2, during the nucleic acid precipitation, the dehydrated alcohol volume that uses is 2～2.5 times of the supernatant volume; Use the Virahol volume to be 0.8～1 times of the supernatant volume.
5. the method for the total RNA of rapid extraction rubus according to claim 1 and 2, it is applicable to raspberry, blackberry, blueberry and other kinds or mutation under the rubus.
6. the method for the total RNA of rapid extraction rubus according to claim 1 and 2, it is characterized in that: described vegetable material comprises fruit, blade and flower.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101638651A (en) * | 2009-07-09 | 2010-02-03 | 昆明理工大学 | Method for extracting total RNA from plant tissue rich in polysaccharides and polyphenols and secondary metabolites |
| CN102191239A (en) * | 2010-03-11 | 2011-09-21 | 中国热带农业科学院橡胶研究所 | Method for extracting total RNA from leechee |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101638651A (en) * | 2009-07-09 | 2010-02-03 | 昆明理工大学 | Method for extracting total RNA from plant tissue rich in polysaccharides and polyphenols and secondary metabolites |
| CN102191239A (en) * | 2010-03-11 | 2011-09-21 | 中国热带农业科学院橡胶研究所 | Method for extracting total RNA from leechee |
Non-Patent Citations (4)
| Title |
|---|
| Chris S.Jones et al..The Isolation of RNA from Raspberry(Rubus idaeus) Fruit.《Biolecular Biotechnology》.1997,第8卷(第3期),219-221. |
| The Isolation of RNA from Raspberry(Rubus idaeus) Fruit;Chris S.Jones et al.;《Biolecular Biotechnology》;19971231;第8卷(第3期);219-221 * |
| 一种从香蕉果实提取高质量RNA的方法;庄军平等;《分子植物育种》;20061231;第4卷(第1期);143-146 * |
| 庄军平等.一种从香蕉果实提取高质量RNA的方法.《分子植物育种》.2006,第4卷(第1期),143-146. |
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