CN102373194B - Method for extracting RNA from tissue of Cunninghamia plant - Google Patents
Method for extracting RNA from tissue of Cunninghamia plant Download PDFInfo
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- CN102373194B CN102373194B CN 201010249646 CN201010249646A CN102373194B CN 102373194 B CN102373194 B CN 102373194B CN 201010249646 CN201010249646 CN 201010249646 CN 201010249646 A CN201010249646 A CN 201010249646A CN 102373194 B CN102373194 B CN 102373194B
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Abstract
The invention discloses a method for extracting RNA from a tissue of a Cunninghamia plant. The method comprises the following steps: 1, cracking the cell wall and the cell membrane of the tissue of the plant, allowing contents in the tissue to release, and removing polyphenol substances and polysaccharide substances; 2, removing proteins on the basis of step 1; 3, removing phenol on the basis of step 2; and 4, depositing RNA on the basis of step 3 to obtain the RNA of the tissue of the plant. The method of the invention, which allows unfavorable influences of the high content of secondary polysaccharide phenol metabolites to the extraction of the RNA in the tissue of the Cunninghamia plant to be overcome and the high quality RNA to be extracted from the tissue, has a large practical application value.
Description
Technical field
The present invention relates to a kind of method of extracting RNA in perennial trees class plant tissue, particularly a kind of method of extracting RNA in the Cunninghamia plant tissue.
Background technology
China fir is one of evergreen coniferous species, is the important fast-growing merchantable timber seeds of China, mainly is distributed in China and south east asia.Growth of Chinese Fir is rapid, and stem is perfectly straight satisfactory, and material is light and tough, beautiful texture, and handling ease, timber fragrance is not forgiven, indeformable, and anti-ant moth is corrosion-resistant.It is widely used in the fields such as building construction, decoration, furniture, shipbuilding, vessel processed.Along with the China fir deep processing utilizes, its purposes is constantly expanded, and adds the raising of people's living standard, requires the living environment back to nature, and China fir more and more is subjected to broad masses welcome and like, especially in southern each province.
For yearly plant, in perennial trees class plant tissue, the secondary metabolic substd content such as polysaccharide polyphenol class is more, and the content of aldehydes matter can increase along with the growth of plant.When vegetable material homogenate, aldehydes matter can discharge, and makes homogenate become brown after oxidation, and deepens with the increase of degree of oxidation, and this phenomenon is called as brownization effect.Oxidized phenolic compound (as quinones) can stably be combined with RNA, thereby affects the separation and purification of RNA, has had a strong impact on the Quality and yield that extracts RNA.When precipitated rna, polysaccharide also often precipitation form spawn, this precipitation is insoluble in water, or produces thick solution after dissolving, so also be difficult to effectively itself and RNA be divided out.Polysaccharide can suppress the activity of many enzymes in addition, and the RNA sample that has therefore polluted polysaccharide can't be used for further molecular biology research.Due to research Cunninghamia growth and development of plants, metabolism and relevant various molecular mechanisms and the forest transgenic research such as degeneration-resistant, all relate to the extraction of RNA in its tissue, but because the practical difficulty of Cunninghamia plant RNA extraction causes Cunninghamia molecular biology of plants and genetically engineered to comprise that forest resistance, reproductive development, Wood properies improvement etc. obviously fall behind.At present, the methods such as the extracting method of RNA such as Trizo1, SDS, conventional CTAB all can not be extracted high-quality RNA effectively from the woody Cunninghamia plant tissue that is rich in polysaccharide polyphenol class material.Also do not extract up to now effective ways and the technology of high quality RNA in the various tissues of Cunninghamia plant, thereby cause the molecular biology research relevant to the Cunninghamia plant and application to fall behind a lot with respect to angiosperm and annual crop.
Summary of the invention
The purpose of this invention is to provide a kind of method of extracting RNA in the Cunninghamia plant tissue.
The method of RNA in extraction Cunninghamia plant tissue provided by the present invention comprises the following steps:
(1) cell walls of cracking plant tissue and cytolemma discharge inclusion and remove polyphenol substance and polysaccharide material;
(2) Deproteinization on the basis of step (1);
(3) phenol that gets on the basis of step (2);
(4) precipitated rna on the basis of step (3) obtains the RNA of described plant tissue.
In described method, in described step (4), after described precipitated rna, comprise the step of removing DNA;
Cell walls and the cytolemma of cracking plant tissue in described step (1), the method that discharges inclusion is: with the RNA Extraction buffer, described plant tissue is carried out extracting, get supernatant liquor;
Described RNA Extraction buffer is comprised of buffer A and beta-mercaptoethanol, the volume ratio 100 of buffer A and beta-mercaptoethanol: 2-100: 8, be specially 100: 2,100: 5 or 100: 8;
Every 1 liter of described buffer A is comprised of 200mL STE damping fluid, 40g sodium laurylsulfonate, 30g polyvinylpyrrolidone, 150mL second alcohol and water, and water is supplied volume, and the pH value is 7.0-8.0; Described water be diethylpyrocarbonate processed without RNA enzyme ultrapure water;
The described STE damping fluid of every 200mL is comprised of 6.0g Tutofusin tris, 8.2g sodium-chlor, 3.7g ethylenediamine tetraacetic acid (EDTA) and water, and water is supplied volume, and the pH value is 8.0; Described water be diethylpyrocarbonate processed without RNA enzyme ultrapure water;
The method of Deproteinization is in described step (2): with the product that the mixed solution extraction steps (1) of phenol, chloroform and primary isoamyl alcohol obtains, collection supernatant liquor;
Go the method for phenol to be in described step (3): with the product that the mixed solution extraction steps (2) of chloroform and primary isoamyl alcohol obtains, to collect supernatant liquor;
In described step (4), the method for precipitated rna is: precipitate or precipitate with Virahol and sodium acetate aqueous solution with dehydrated alcohol and sodium acetate aqueous solution.
3, method according to claim 1 and 2 is characterized in that:
In described step (1), describedly with the RNA Extraction buffer to the method that described plant tissue carries out extracting be: with described RNA Extraction buffer and described plant tissue mixing, place 5min-10min or 5min or 7min or 10min, more centrifugal, get supernatant liquor;
In described step (2), the product that obtains with the mixed solution extraction steps (1) of phenol, chloroform and primary isoamyl alcohol, the method of collecting supernatant liquor is: the product that mixed solution and the step (1) of described phenol, chloroform and primary isoamyl alcohol obtained was with the volume ratio mixing of 1: 1, place 5min-10min or 5min or 7min or 10min, centrifugal again, collect supernatant liquor;
In described step (3), the product that obtains with the mixed solution extraction steps (2) of chloroform and primary isoamyl alcohol, the method of collecting supernatant liquor is: the product that step (2) is obtained and the mixed solution of chloroform and primary isoamyl alcohol were with the volume ratio mixing of 1: 1, place 5min-10min or 5min or 7min or 10min, centrifugal, collect supernatant liquor;
In described step (4), the method that precipitates with dehydrated alcohol and sodium acetate aqueous solution is: the product that step (3) is obtained mixes with dehydrated alcohol and sodium acetate aqueous solution, place 30min-120min or 30min or 75min or 120min for-20 ℃, more centrifugal, collecting precipitation; The method that precipitates with Virahol and sodium acetate aqueous solution is: the product that step (3) is obtained mixes with Virahol and sodium acetate aqueous solution, places 30min-120min or 30min or 75min or 120min for-20 ℃, more centrifugal, collecting precipitation; The product that described step (3) obtains and the volume ratio of dehydrated alcohol and sodium acetate aqueous solution are 10: 25: 1; The product that described step (3) obtains and the volume ratio of Virahol and sodium acetate aqueous solution are 10: 10: 1.
4, arbitrary described method according to claim 1-3 is characterized in that:
In described step (1), the proportioning of described RNA Extraction buffer and described plant tissue is the 0.25g-1g plant tissue: 4mL RNA Extraction buffer is specially the 0.25g plant tissue: 4mL RNA Extraction buffer, 0.6g plant tissue: 4mL RNA Extraction buffer or 1g plant tissue: 4mL RNA Extraction buffer;
In described step (4), the concentration of described sodium acetate aqueous solution is 3mol/L.
In described step (1), described centrifugal mode is that rotation radius is 66mm at 4 ℃ of centrifugal 5min of the speed with 12,000r/min;
In described step (2), described centrifugal mode is that rotation radius is 66mm at 4 ℃ of centrifugal 15min of the speed with 12,000r/min;
In described step (3), described centrifugal mode is that rotation radius is 66mm at 4 ℃ of centrifugal 5min of the speed with 12,000r/min;
In described step (4), described centrifugal mode is that rotation radius is 66mm at 4 ℃ of centrifugal 10min of the speed with 12,000r/min.
In described step (2), in the mixed solution of described phenol, chloroform and primary isoamyl alcohol, the volume ratio of phenol, chloroform and primary isoamyl alcohol is 25: 24: 1;
In described step (3), in the mixed solution of described chloroform and primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol is 24: 1.
In described step (1), described plant tissue is the plant tissue powder.
Described plant is the Cunninghamia plant; Described Cunninghamia plant is China fir; Described plant tissue is leaf or stem.
In method of the present invention, before extracting, adopt strict processing without RNase, add SDS can make the rapid sex change of albumen in Extraction buffer, Polysaccharide removing, beta-mercaptoethanol can separate interference and the destruction that produces, the polysaccharide polyphenol class material of being combined with PVP by the Polysaccharide removing polyphenols to RNA because of oxidation, can directly get rid of by centrifugal, or remove when chloroform/primary isoamyl alcohol extracting.Phenol/chloroform/primary isoamyl alcohol extracting, the pollution that can effectively remove albumen and polysaccharide.DNase I without RNase processes RNA solution, and the pollution that can effectively remove the genomic dna in RNA obtains high purity RNA.It is high to total RNA extraction adverse effect in the Cunninghamia plant tissue that the present invention has effectively overcome polysaccharide polyphenol class secondary metabolites content, can obtain high-quality RNA from wherein extracting, the total RNA purity that obtains is high, the pollution of effectively having removed albumen, salt and polysaccharide and Polyphenols secondary metabolites.Therefore, the inventive method has larger actual application value.
Description of drawings
Fig. 1 is that the China fir needle organizes the RNA of genomic dna through 1% agarose gel electrophoresis figure.
Fig. 2 is that China fir needle tissue is removed the RNA of genomic dna through 1% agarose gel electrophoresis figure.
Fig. 3 is that the China fir stem organizes the RNA of genomic dna through 1% agarose gel electrophoresis figure.
Fig. 4 is that China fir stem tissue is removed the RNA of genomic dna through 1% agarose gel electrophoresis figure.
Embodiment
The experimental technique that uses in following embodiment is ordinary method if no special instructions.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Lignum seu Ramulus Cunninghamiae Lanceolatae leaf is available from the new nursery of good fortune, Sanming City, Fujian Province Qingliu County; The China fir stem is available from the new nursery of good fortune, Sanming City, Fujian Province Qingliu County.
Embodiment 1, extraction China fir needle are organized RNA (not removing DNA) and detect
Method I
One, extract the China fir needle and organize RNA (not removing DNA)
All solution, container and utensil etc. are strictly undertaken processing without the RNase method by the described method of molecular cloning second edition.
Extracting the China fir needle organizes RNA (not removing DNA) to comprise the following steps:
1, the cell walls of cracking plant tissue and cytolemma discharge inclusion and remove polyphenol substance and polysaccharide material RNA Extraction buffer is comprised of buffer A and beta-mercaptoethanol;
Every 1 liter of described buffer A is comprised of 200mL STE damping fluid, 40g sodium laurylsulfonate (SDS), 30g polyvinylpyrrolidone, 150mL second alcohol and water, water is supplied volume (water is the ultrapure water without the RNA enzyme that diethylpyrocarbonate (DEPC) is processed), and the pH value is 7.0;
The described STE damping fluid of every 200mL is comprised of 6.0g Tutofusin tris (Tris), 8.2g sodium-chlor (NaCl), 3.7g ethylenediamine tetraacetic acid (EDTA) (EDTA) and water, water is supplied volume (water is the ultrapure water without the RNA enzyme that DEPC processes), and the pH value is 8.0;
Before use, add beta-mercaptoethanol in buffer A, obtain the RNA Extraction buffer; The addition of beta-mercaptoethanol is 50mL beta-mercaptoethanol/1 liter buffer A;
Grind 200mg China fir needle to Powdered in liquid nitrogen, change over to rapidly in the 1.5mL centrifuge tube of precooling, according to the 0.25g plant tissue: the ratio of 4mL RNA Extraction buffer adds above-mentioned RNA Extraction buffer, and vortex is 30 seconds rapidly, and room temperature is placed 5min; At 4 ℃ of centrifugal 5min of the speed with 12,000r/min, rotation radius is 66mm, collects supernatant liquor and forwards in another centrifuge tube.Contain SDS, NaCl and ethanol in the RNA Extraction buffer, but by high speed centrifugation Polysaccharide removing and polysaccharide derivates.
2, Deproteinization on the basis of step (1)
The product that the mixed solution (volume ratio of phenol, chloroform and primary isoamyl alcohol is 25: 24: 1) of phenol, chloroform and primary isoamyl alcohol and step (1) are obtained was with the volume ratio mixing of 1: 1, place 5min, at 4 ℃ of centrifugal 15min of the speed with 12,000r/min, rotation radius is 66mm; Collecting supernatant liquor forwards in another centrifuge tube.
3, the phenol that gets on the basis of step (2)
The product that the mixed solution (volume ratio of chloroform and primary isoamyl alcohol is 24: 1) of chloroform and primary isoamyl alcohol and step (2) are obtained is placed 5min with the volume ratio mixing of 1: 1, and at 4 ℃ of centrifugal 5min of the speed with 12,000r/min, rotation radius is 66mm; Collecting supernatant liquor forwards in another centrifuge tube.
4, precipitated rna on the basis of step (3) obtains the RNA of plant tissue.
The product that step (3) is obtained mixed as 10: 25: 1 take volume ratio with dehydrated alcohol and sodium acetate aqueous solution (3mol/L), placed 30min for-20 ℃, and at 4 ℃ of centrifugal 10min of the speed with 12,000r/min, rotation radius is 66mm; Collecting precipitation obtains the China fir needle and organizes RNA;
Precipitation is washed one time with 1mL70% ethanol, at 4 ℃ of centrifugal 5min of the speed with 12,000r/min, removes supernatant liquor fully, collecting precipitation; To be deposited in air drying 5min, add the water dissolution precipitation that 50 μ L DEPC process, obtain RNA solution, be kept in-80 ℃ of Ultralow Temperature Freezers.
The compound method of 3mol/L sodium acetate aqueous solution: 24.6 gram sodium-acetates are dissolved in the H that DEPC processes
2In O, regulate pH to 5.2, be settled to 100mL, autoclaving.
Two, detect
1, the integrity of electrophoresis detection RNA
Get the China fir needle that above-mentioned steps one obtains and organize RNA 1 μ L, electrophoresis on 1% sepharose, 180V, electrophoresis 10-20min, it the results are shown in Figure 1.As can be seen from the figure, organize RNA (not removing DNA) through the China fir needle that method of the present invention is extracted, electrophoresis detection 28S and 18S band are all very clear, and ratio is 2: 1, illustrates that RNA has kept integrity preferably.But through removing the step of DNA, the RNA that obtains does not still have the pollution of genomic dna.
2, extract the purity detecting of RNA
Get the China fir needle that 2 μ L step 1 obtain and organize the RNA sample, with 50 times of 1 * TE (pH8.0) dilutions, do blank with 100 μ L 1 * TE (pH8.0), the reading of regulating ultraviolet spectrophotometer at 230nm, 260nm, 280nm place is to zero, then reads testing sample in the OD value at three wavelength places.Result shows: ultraviolet spectrophotometer records OD
230Be 0.251, OD
260Be 0.512, OD
280Be 0.261, wherein, OD
260/ OD
280=1.962, OD
260/ OD
230=2.040.OD
260/ OD
280Ratio shows that RNA purity is high between 1.9-2.1, there is no remaining protein contamination, OD
260/ OD
230Greater than 2.0, the pollution that there is no impurity salt is described.
Method II
One, extract the China fir needle and organize RNA (not removing DNA)
All solution, container and utensil etc. are strictly undertaken processing without the RNase method by the described method of molecular cloning second edition.
Extracting the China fir needle organizes RNA (not removing DNA) to comprise the following steps:
1, the cell walls of cracking plant tissue and cytolemma, discharge inclusion and remove polyphenol substance and polysaccharide material except following 1)-3), all the other methods are all identical with method I
(1) in the RNA Extraction buffer, the pH value of buffer A is 7.5;
(2) before using the RNA Extraction buffer, add beta-mercaptoethanol in buffer A, the addition of beta-mercaptoethanol is 20mL beta-mercaptoethanol/1 liter buffer A;
(3) according to the 0.6g plant tissue: the ratio of 4mL RNA Extraction buffer adds above-mentioned RNA Extraction buffer, and vortex is 45 seconds rapidly, and room temperature is placed 7min.
2, Deproteinization on the basis of step (1)
Except the volume ratio mixing of the product that mixed solution and the step (1) of phenol, chloroform and primary isoamyl alcohol obtained with 1: 1, beyond placement 7min, all the other methods are identical with method I.
3, the phenol that gets on the basis of step (2)
Except the volume ratio mixing of the product that mixed solution and the step (2) of chloroform and primary isoamyl alcohol obtained with 1: 1, beyond placement 7min, all the other methods are identical with method I.
4, precipitated rna on the basis of step (3) obtains the RNA of plant tissue.
Except the product that step (3) is obtained mixed as 10: 10: 1 take volume ratio with Virahol and sodium acetate aqueous solution (3mol/L), to place outside 75min for-20 ℃, all the other methods are all identical with method I.
Two, detect
1, the integrity of electrophoresis detection RNA
With method I result without significant difference.
2, extract the purity detecting of RNA
With method I result without significant difference.
Method III
One, extract the China fir needle and organize RNA (not removing DNA)
All solution, container and utensil etc. are strictly undertaken processing without the RNase method by the described method of molecular cloning second edition.
Extracting the China fir needle organizes RNA (not removing DNA) to comprise the following steps:
1, the cell walls of cracking plant tissue and cytolemma, discharge inclusion and remove polyphenol substance and polysaccharide material except following 1)-3), all the other methods are all identical with method I
(1) in the RNA Extraction buffer, the pH value of buffer A is 8.0;
(2) before using the RNA Extraction buffer, add beta-mercaptoethanol in buffer A, the addition of beta-mercaptoethanol is 80mL beta-mercaptoethanol/1 liter buffer A;
(3) according to the 1g plant tissue; The ratio of 4mL RNA Extraction buffer adds above-mentioned RNA Extraction buffer, and vortex is 60 seconds rapidly, and room temperature is placed 10min.
2, Deproteinization on the basis of step (1)
Except the volume ratio mixing of the product that mixed solution and the step (1) of phenol, chloroform and primary isoamyl alcohol obtained with 1: 1, beyond placement 10min, all the other methods are identical with method I.
3, the phenol that gets on the basis of step (2)
Except the volume ratio mixing of the product that mixed solution and the step (2) of chloroform and primary isoamyl alcohol obtained with 1: 1, beyond placement 10min, all the other methods are identical with method I.
4, precipitated rna on the basis of step (3) obtains the RNA of plant tissue
With after dehydrated alcohol and sodium acetate aqueous solution mix, outside-20 ℃ of placement 120min, all the other methods are all identical with method I except the product that step (3) is obtained.
Two, detect
1, the integrity of electrophoresis detection RNA
With method I result without significant difference.
2, extract the purity detecting of RNA
With method I result without significant difference.
Embodiment 2, extraction China fir needle are organized RNA (removing DNA)
One, extract the China fir needle and organize RNA (removing DNA)
It is in embodiment 1 step (4) back that extraction China fir needle is organized RNA (removing DNA), increases the step of removing DNA, and the method I of other step and embodiment 1 is identical.The step of removing DNA comprises as follows:
(a) use without the genomic dna (10 * DNase I buffer, the 10U DNase I, 20U RNase inhibitor, the 20-50 μ L RNA solution that contain 5 μ L in 50 μ L reaction systems) in the DNase I removal RNA of RNase, 37 ℃ of reaction 30min;
(b) product of chloroform/primary isoamyl alcohol (24: 1) with isopyknic step (a) mixed, standing 5-10min, 12,000r/min, 4 ℃ of centrifugal 10min collect supernatant liquor;
(c) precipitated rna: intermediate processing is identical with the step (4) of the method I of embodiment 1;
(d) wash precipitation with 1mL 70% ethanol, 4 ℃, the centrifugal 5min of 12,000r/min removes supernatant fully;
(e) be deposited in air drying 5min, add the water dissolution RNA precipitation that 50 μ L DEPC process, in-80 ℃ of preservations.
Two, detect
1, the integrity of electrophoresis detection RNA
Get the China fir needle that above-mentioned steps one obtains and organize RNA 1 μ L, electrophoresis on 1% sepharose, 180V, electrophoresis 10-20min, it the results are shown in Figure 2.As can be seen from the figure, organize RNA (removing DNA) through the China fir needle that method of the present invention is extracted, electrophoresis detection 28S and 18S band are all very clear, and ratio is 2: 1, illustrates that RNA has kept integrity preferably.
2, extract the purity detecting of RNA
Get the China fir needle that 2 μ L step 1 obtain and organize the RNA sample, with 50 times of 1 * TE (pH8.0) dilutions, do blank with 100 μ L 1 * TE (pH8.0), the reading of regulating ultraviolet spectrophotometer at 230nm, 260nm, 280nm place is to zero, then reads testing sample in the OD value at three wavelength places.Result shows: ultraviolet spectrophotometer records OD
230Be 0.151, OD
260Be 0.338, OD
280Be 0.162, wherein, OD
260/ OD
280=2.086, OD
260/ OD
230=2.238.OD
260/ OD
280Ratio shows that RNA purity is high between 1.9-2.1, there is no remaining protein contamination, OD
260/ OD
230Greater than 2.0, the pollution that there is no impurity salt is described.
Embodiment 3, extraction China fir stem are organized RNA (not removing DNA)
One, extract the China fir stem and organize RNA (not removing DNA)
In this embodiment, material used is China fir stem tissue, and the RNA extracting method of this embodiment is identical with RNA extracting method in the method I of embodiment 1.
Two, detect
1, the integrity of electrophoresis detection RNA
Get the China fir stem that above-mentioned steps one obtains and organize RNA 1 μ L, electrophoresis on 1% sepharose, 180V, electrophoresis 10-20min, it the results are shown in Figure 3.As can be seen from the figure, organize RNA (not removing DNA) through the China fir needle that method of the present invention is extracted, electrophoresis detection 28S and 18S band are all very clear, and ratio is 2: 1, illustrates that RNA has kept integrity preferably.But through removing the step of DNA, the RNA that obtains does not still have the pollution of genomic dna.
2, extract the purity detecting of RNA
Get the China fir stem that 2 μ L step 1 obtain and organize the RNA sample, with 50 times of 1 * TE (pH8.0) dilutions, do blank with 100 μ L1 * TE (pH8.0), the reading of regulating ultraviolet spectrophotometer at 230nm, 260nm, 280nm place is to zero, then reads testing sample in the OD value at three wavelength places.Result shows: ultraviolet spectrophotometer records OD
230Be 0.331, OD
260Be 0.678, OD
280Be 0.346, wherein, OD
260/ OD
280=1.960, OD
260/ OD
230=2.048.OD
260/ OD
280Ratio shows that RNA purity is high between 1.9-2.1, there is no remaining protein contamination, OD
260/ OD
230Greater than 2.0, the pollution that there is no impurity salt is described.
Embodiment 4, extraction China fir stem are organized RNA (removing DNA)
One, extract the China fir stem and organize RNA (removing DNA)
In this embodiment, material used is China fir stem tissue, and the RNA extracting method of this embodiment is identical with the RNA extracting method of embodiment 2.
Two, detect
1, the integrity of electrophoresis detection RNA
Get the China fir stem that above-mentioned steps one obtains and organize RNA 1 μ L, electrophoresis on 1% sepharose, 180V, electrophoresis 10-20min, it the results are shown in Figure 4.As can be seen from the figure, organize RNA (not removing DNA) through the China fir needle that method of the present invention is extracted, electrophoresis detection 28S and 18S band are all very clear, and ratio is 2: 1, illustrates that RNA has kept integrity preferably.
2, extract the purity detecting of RNA
Get the China fir stem that 2 μ L step 1 obtain and organize the RNA sample, with 50 times of 1 * TE (pH8.0) dilutions, do blank with 100 μ L1 * TE (pH8.0), the reading of regulating ultraviolet spectrophotometer at 230nm, 260nm, 280nm place is to zero, then reads testing sample in the OD value at three wavelength places.Result shows: ultraviolet spectrophotometer records OD
230Be 0.203, OD
260Be 0.428, OD
280Be 0.212, wherein, OD
260/ OD
280=2.019, OD
260/ OD
230=2.108.OD
260/ OD
280Ratio shows that RNA purity is high between 1.9-2.1, there is no remaining protein contamination, OD
260/ OD
230Greater than 2.0, the pollution that there is no impurity salt is described.
Claims (6)
1. method of extracting plant tissue RNA comprises the following steps:
(1) cell walls of cracking plant tissue and cytolemma discharge inclusion and remove polyphenol substance and polysaccharide material;
(2) Deproteinization on the basis of step (1);
(3) phenol that gets on the basis of step (2);
(4) precipitated rna on the basis of step (3) obtains the RNA of described plant tissue;
In described method, in described step (4), after described precipitated rna, comprise the step of removing DNA;
Cell walls and the cytolemma of cracking plant tissue in described step (1), the method that discharges inclusion is: with the RNA Extraction buffer, described plant tissue is carried out extracting, get supernatant liquor;
Described RNA Extraction buffer is comprised of buffer A and beta-mercaptoethanol, the volume ratio 100 of buffer A and beta-mercaptoethanol: 2-100: 8;
Every 1 liter of described buffer A is comprised of 200mL STE damping fluid, 40g sodium laurylsulfonate, 30g polyvinylpyrrolidone, 150mL second alcohol and water, and water is supplied volume, and the pH value is 7.0-8.0;
The described STE damping fluid of every 200mL is comprised of 6.0g Tutofusin tris, 8.2g sodium-chlor, 3.7g ethylenediamine tetraacetic acid (EDTA) and water, and water is supplied volume, and the pH value is 8.0;
The method of Deproteinization is in described step (2): with the product that the mixed solution extraction steps (1) of phenol, chloroform and primary isoamyl alcohol obtains, collection supernatant liquor;
Go the method for phenol to be in described step (3): with the product that the mixed solution extraction steps (2) of chloroform and primary isoamyl alcohol obtains, to collect supernatant liquor;
In described step (4), the method for precipitated rna is: precipitate or precipitate with Virahol and sodium acetate aqueous solution with dehydrated alcohol and sodium acetate aqueous solution;
Described plant is China fir; Described plant tissue is leaf or stem.
2. method according to claim 1 is characterized in that:
In described step (1), describedly with the RNA Extraction buffer to the method that described plant tissue carries out extracting be: with described RNA Extraction buffer and described plant tissue mixing, place 5min-10min, more centrifugal, get supernatant liquor;
In described step (2), the product that obtains with the mixed solution extraction steps (1) of phenol, chloroform and primary isoamyl alcohol, the method of collecting supernatant liquor is: the product that mixed solution and the step (1) of described phenol, chloroform and primary isoamyl alcohol obtained was with the volume ratio mixing of 1: 1, place 5min-10min, centrifugal again, collect supernatant liquor;
In described step (3), the product that obtains with the mixed solution extraction steps (2) of chloroform and primary isoamyl alcohol, the method of collecting supernatant liquor is: the product that step (2) is obtained and the mixed solution of chloroform and primary isoamyl alcohol were with the volume ratio mixing of 1: 1, place 5min-10min, centrifugal, collect supernatant liquor;
In described step (4), the method that precipitates with dehydrated alcohol and sodium acetate aqueous solution is: the product that step (3) is obtained mixes with dehydrated alcohol and sodium acetate aqueous solution, places 30min-120min for-20 ℃, more centrifugal, collecting precipitation; The method that precipitates with Virahol and sodium acetate aqueous solution is: the product that step (3) is obtained mixes with Virahol and sodium acetate aqueous solution, places 30min-120min for-20 ℃, more centrifugal, collecting precipitation; The product that described step (3) obtains and the volume ratio of dehydrated alcohol and sodium acetate aqueous solution are 10: 25: 1; The product that described step (3) obtains and the volume ratio of Virahol and sodium acetate aqueous solution are 10: 10: 1.
3. method according to claim 1 and 2 is characterized in that:
In described step (1), the proportioning of described RNA Extraction buffer and described plant tissue is the 0.25g-1g plant tissue: 4mL RNA Extraction buffer;
In described step (4), the concentration of described sodium acetate aqueous solution is 3mol/L.
4. method according to claim 1 is characterized in that:
In described step (1), described centrifugal mode is that rotation radius is 66mm at 4 ℃ of centrifugal 5min of the speed with 12,000r/min;
In described step (2), described centrifugal mode is that rotation radius is 66mm at 4 ℃ of centrifugal 15min of the speed with 12,000r/min;
In described step (3), described centrifugal mode is that rotation radius is 66mm at 4 ℃ of centrifugal 5min of the speed with 12,000r/min;
In described step (4), described centrifugal mode is that rotation radius is 66mm at 4 ℃ of centrifugal 10min of the speed with 12,000r/min.
5. method according to claim 1 is characterized in that:
In described step (2), in the mixed solution of described phenol, chloroform and primary isoamyl alcohol, the volume ratio of phenol, chloroform and primary isoamyl alcohol is 25: 24: 1;
In described step (3), in the mixed solution of described chloroform and primary isoamyl alcohol, the volume ratio of chloroform and primary isoamyl alcohol is 24: 1.
6. method according to claim 1, it is characterized in that: in described step (1), described plant tissue is the plant tissue powder.
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