CN102899319B - Method for extracting RNA (Ribonucleic Acid) from kiwi fruit leaf - Google Patents
Method for extracting RNA (Ribonucleic Acid) from kiwi fruit leaf Download PDFInfo
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- CN102899319B CN102899319B CN201210449428.5A CN201210449428A CN102899319B CN 102899319 B CN102899319 B CN 102899319B CN 201210449428 A CN201210449428 A CN 201210449428A CN 102899319 B CN102899319 B CN 102899319B
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Abstract
The invention discloses a method for extracting RNA (Ribonucleic Acid) from kiwi fruit leaves. The method comprises the following steps of: (1) pre-processing the kiwi fruit leaves, eliminating the polysaccharide and polyphenols impurities in the kiwi fruit leaves by using an alcohol solution of guanidinium isothiocyanate so as to prevent the influence of the polysaccharide and polyphenols impurities on the extraction of the RNA of the kiwi fruit; (2) extracting the total RNA in the kiwi fruit leaves; and (3) purifying the RNA of the kiwi fruit. By utilizing the method for extracting the RNA in the kiwi fruit leaves, the influence of the polysaccharide and the polyphenols impurities in the leaves on the extraction of the RNA of the kiwi fruit can be effectively eliminated, so that the extracted RNA of the kiwi fruit is high in purity and good in quality, and better completeness is maintained.
Description
Technical field
The present invention relates to the extracting method of a kind of RNA, specifically refer to extract the novel method of RNA from Kiwifruit blade.
Background technology
The quality that in Kiwifruit blade, total RNA extracts quality is one of key of the synthetic cDNA success or failure of Kiwifruit transgenic breeding reverse transcription, by extracting the RNA in Kiwifruit blade and can obtaining the transgenosis Kiwifruit new variety of various characteristics in conjunction with other biotechnology, make the extraction of RNA in Kiwifruit blade there is significant application value.
In Kiwifruit blade, the content of soluble polysaccharide, polyphenol is higher, and many physico-chemical properties of polysaccharide, polyphenol are similar with RNA, easy and RNA forms the jelly co-precipitation of indissoluble, cause RNA output to reduce, or after dissolving, polysaccharide, polyphenol form sticky contaminated aqueous solution RNA sample, cause RNA quality to reduce, thereby affect the breeding of transgenosis Kiwifruit.
In prior art, Trizol method is the universal method that majority of plant RNA extracts, but be not suitable for the extraction of the polysaccharide polyphenol class plant RNAs such as Kiwifruit, while extracting in Kiwifruit blade RNA by Trizol method, sample is very fast brownization in Trizol damping fluid, supernatant liquor agglutination when with chloroform extracting, and the impact of polysaccharide polyphenol is very serious, RNA is difficult to separated, causes that RNA purity is low, poor quality.
Therefore, the new method for extracting of RNA in exploitation Kiwifruit blade, complete, the high purity that guarantees in monkey peach leaf sheet that RNA extracts and higher extraction yield are significant.
Summary of the invention
For solving the technical problem that in Kiwifruit blade, RNA extracts, the inventor passes through lot of experiments, finally invent a kind of novel method of extracting RNA from Kiwifruit blade, use the method, complete, the high purity that can guarantee in monkey peach leaf sheet that RNA extracts and higher extraction yield.
The present invention includes following step:
(1) pre-treatment of Kiwifruit blade.After the Kiwifruit blade of just having won is shredded, drop into immediately massfraction and be in the ethanolic soln of 50%-80% of guanidinium isothiocyanate of 2%-4%, the mass volume ratio of Kiwifruit blade and solution be 1:50-1:200(quality unit for gram, the unit of volume is milliliter), lixiviate 1-3 hour under normal temperature.
(2) extraction of the total RNA of Kiwifruit.By RNA Extraction buffer incubation 30-40 minutes in 55-75 ℃ of water-bath, the blade of getting immediately above-mentioned processing grinds in mortar fast with damping fluid that (mass volume ratio of blade and damping fluid is 1:10-1:30, the unit of quality is gram, the unit of volume is milliliter, adds liquid nitrogen to prevent RNA degraded in process of lapping).Ground, become thick homogenate to move in centrifuge tube, 55-75 ℃ of incubation 5-15 minute, (volume ratio of chloroform and primary isoamyl alcohol is 20: 1-30:1) to add the mixing solutions of isopyknic chloroform and primary isoamyl alcohol, upper and lower concuss 10-90 second, then in 0-20 ℃, 5000-20000 g, centrifugal 5-60 minute.Get supernatant, repeat above-mentioned extraction steps 2-4 time.Get supernatant, add the 5-15M LiCl solution of supernatant liquor 1/5-1/2 volume and mix ,-10 ℃--under 50 ℃ of conditions, place 10-20 hour, make RNA precipitation.0-20 ℃, 5000-20000 g, centrifugal 5-60 minute, abandons supernatant, obtains total RNA precipitation of slightly putting forward.
RNA Extraction buffer is a kind of aqueous solution, in this solution, the massfraction of CTAB is 2%,, PVP40 massfraction be that the concentration of 2%-5%, Tris-Cl is 100 mM(pH 8.0), concentration 25 mM(pH 8.0 of EDTA), the concentration of NaCl is 2 M, and the massfraction of β-ME is 2-5% (used time adds).
(3) purifying of Kiwifruit RNA.Adding DEPC treated water to dissolve total RNA(water obtained above is 5:1-20:1 with the volume mass ratio of total RNA, the unit of volume is ml, the unit of quality is g), then add the 1-10 M NaAc of DEPC treated water 1/30-1/3 volume, the ethanol of 90 %-98% of 1-10 volume.Be placed in-5 ℃--50 ℃, 1-5 hour, makes RNA precipitation.0-20 ℃, 5000-20000 g, centrifugal 5-60 minute, abandons supernatant, by 50%-95% ethanol washing and precipitating, obtains RNA after air drying.(RNA obtaining can add DEPC treated water to dissolve, be stored in-70 ℃ standby or directly with anhydrous alcohol solution, preserve).
In aforesaid operations process, all vessel are all processed through killing RNA enzymic activity, and the RNA extracting is not destroyed; Related ethanol concn, refers to volume ratio.The Kiwifruit RNA obtaining by the present invention detects through 1% agarose gel electrophoresis, see two bands clearly, and two band luminance factors are 1.8:1-2.2:1, ultraviolet spectrophotometer is measured A260nm and A280nm ratio, ratio, between 1.9-2.0, is high-quality RNA.
Feature of the present invention is to take Kiwifruit blade as material, with guanidinium isothiocyanate spirituous solution, first removes polysaccharide, the Polyphenols impurity in blade, and the impact of avoiding polysaccharide, polyphenols to extract Kiwifruit RNA makes that the Kiwifruit RNA purity extracted is high, quality better; The integrity that has also kept RNA when removing polysaccharide, Polyphenols impurity in blade with guanidinium isothiocyanate spirituous solution.
Embodiment
Below the concrete extracting method of RNA in Kiwifruit blade is elaborated.
Embodiment 1:
(1) pre-treatment of Kiwifruit blade.After the Kiwifruit blade of just having won is shredded, drop into immediately massfraction and be in 55% ethanolic soln of 2.2% guanidinium isothiocyanate, the mass volume ratio of Kiwifruit blade and solution is 1:60, and under normal temperature, lixiviate is 1.5 hours.
(2) extraction of the total RNA of Kiwifruit.By RNA Extraction buffer incubation 32 minutes in 65 ℃ of water-baths, the blade of getting immediately above-mentioned processing grinds fast (mass volume ratio of blade and damping fluid is 1:15) in mortar with damping fluid.Ground, become thick homogenate to move in centrifuge tube, 65 ℃ of incubations 10 minutes, the mixing solutions (in mixing solutions, the volume ratio of chloroform and primary isoamyl alcohol is 24: 1) that adds isopyknic chloroform and primary isoamyl alcohol, upper and lower concuss 20 seconds, then in 4 ℃, 12000 g, centrifugal 10 minutes.Get supernatant, repeat above-mentioned extraction steps 2 times.Get supernatant, add the 8M LiCl solution of supernatant liquor 1/4 volume and mix, under-20 ℃ of conditions, place 12 hours, make RNA precipitation.4 ℃, 12000 g, centrifugal 10 minutes, abandon supernatant, obtain total RNA precipitation of slightly putting forward.
(3) purifying of Kiwifruit RNA.Add volume mass that DEPC treated water dissolves total RNA(water obtained above and total RNA than being 10:1), then add 5 M NaAc of DEPC treated water 1/10 volume, 95% ethanol of 5 volumes.Be placed in-20 ℃, 2 hours, make RNA precipitation.4 ℃, 12000 g, centrifugal 10 minutes, abandon supernatant, by 80% ethanol washing and precipitating, after air drying, obtain RNA.
The Kiwifruit RNA obtaining by aforesaid method detects through 1% agarose gel electrophoresis, sees two bands clearly, and two band luminance factors are 1.9:1, and ultraviolet spectrophotometer is measured A260nm and A280nm ratio, and ratio is 1.92.
Embodiment 2:
(1) pre-treatment of Kiwifruit blade.After the Kiwifruit blade of just having won is shredded, drop into immediately massfraction and be in 75% ethanolic soln of 3.6% guanidinium isothiocyanate, the mass volume ratio of Kiwifruit blade and solution is 1:150, and under normal temperature, lixiviate is 2.5 hours.
(2) extraction of the total RNA of Kiwifruit.By RNA Extraction buffer incubation 37 minutes in 60 ℃ of water-baths, the blade of getting immediately above-mentioned processing grinds fast (mass volume ratio of blade and damping fluid is 1:26) in mortar with damping fluid.Ground, become thick homogenate to move in centrifuge tube, 60 ℃ of incubations 14 minutes, the mixing solutions (in mixing solutions, the volume ratio of chloroform and primary isoamyl alcohol is 29: 1) that adds isopyknic chloroform and primary isoamyl alcohol, upper and lower concuss 60 seconds, then in 3 ℃, 11000 g, centrifugal 15 minutes.Get supernatant, repeat above-mentioned extraction steps 3 times.Get supernatant, add the 12M LiCl solution of supernatant liquor 1/3 volume and mix, under-30 ℃ of conditions, place 12 hours, make RNA precipitation.3 ℃, 11000 g, centrifugal 15 minutes, abandon supernatant, obtain total RNA precipitation of slightly putting forward.
(3) purifying of Kiwifruit RNA.Add volume mass that DEPC treated water dissolves total RNA(water obtained above and total RNA than being 17:1), then add 6 M NaAc of DEPC treated water 1/5 volume, 91% ethanol of 8 volumes.Be placed in-25 ℃, 1.8 hours, make RNA precipitation.3 ℃, 11000 g, centrifugal 15 minutes, abandon supernatant, by 88% ethanol washing and precipitating, after air drying, obtain RNA.
The Kiwifruit RNA obtaining by aforesaid method detects through 1% agarose gel electrophoresis, sees two bands clearly, and two band luminance factors are 2.1:1, and ultraviolet spectrophotometer is measured A260nm and A280nm ratio, and ratio is 1.96.
Embodiment 3:
(1) pre-treatment of Kiwifruit blade.After the Kiwifruit blade of just having won is shredded, drop into immediately massfraction and be in 60% ethanolic soln of 3% guanidinium isothiocyanate, the mass volume ratio of Kiwifruit blade and solution is 1:100, and under normal temperature, lixiviate is 2 hours.
(2) extraction of the total RNA of Kiwifruit.By RNA Extraction buffer incubation 35 minutes in 70 ℃ of water-baths, the blade of getting immediately above-mentioned processing grinds fast (mass volume ratio of blade and damping fluid is 1:15) in mortar with damping fluid.Ground, become thick homogenate to move in centrifuge tube, 70 ℃ of incubations 10 minutes, the mixing solutions (in mixing solutions, the volume ratio of chloroform and primary isoamyl alcohol is 21: 1) that adds isopyknic chloroform and primary isoamyl alcohol, upper and lower concuss 30 seconds, then in 5 ℃, 13000 g, centrifugal 10 minutes.Get supernatant, repeat above-mentioned extraction steps 3 times.Get supernatant, add the 7M LiCl solution of supernatant liquor 1/4 volume and mix, under-20 ℃ of conditions, place 12 hours, make RNA precipitation.5 ℃, 13000 g, centrifugal 10 minutes, abandon supernatant, obtain total RNA precipitation of slightly putting forward.
(3) purifying of Kiwifruit RNA.Add volume mass that DEPC treated water dissolves total RNA(water obtained above and total RNA than being 10:1), then add 9 M NaAc of DEPC treated water 1/6 volume, 91% ethanol of 7 volumes.Be placed in-25 ℃, 3.8 hours, make RNA precipitation.5 ℃, 13000 g, centrifugal 10 minutes, abandon supernatant, by 78% ethanol washing and precipitating, after air drying, obtain RNA.
The Kiwifruit RNA obtaining by aforesaid method detects through 1% agarose gel electrophoresis, sees two bands clearly, and two band luminance factors are 2:1, and ultraviolet spectrophotometer is measured A260nm and A280nm ratio, and ratio is 1.93.
Claims (1)
1. an extracting method of RNA in Kiwifruit blade, is characterized in that comprising following step:
(1) pre-treatment of Kiwifruit blade; After the Kiwifruit blade of just having won is shredded, drop into immediately massfraction and be in the ethanolic soln of 50%-80% of guanidinium isothiocyanate of 2%-4%, the mass volume ratio of Kiwifruit blade and solution is 1:50-1:200, lixiviate 1-3 hour under normal temperature;
(2) extraction of the total RNA of Kiwifruit; By RNA Extraction buffer incubation 30-40 minutes in 55-75 ℃ of water-bath, the blade of getting immediately above-mentioned processing adds RNA Extraction buffer and grinds fast in mortar, and the mass volume ratio of its Leaf and RNA Extraction buffer is 1:10-1:30; Ground, become thick homogenate to move in centrifuge tube, 55-75 ℃ of incubation 5-15 minute, the mixing solutions that adds isopyknic chloroform and primary isoamyl alcohol, wherein in mixing solutions, the volume ratio of chloroform and primary isoamyl alcohol is 20: 1-30:1, upper and lower concuss 10-90 second, then in 0-20 ℃, 5000-20000 g, centrifugal 5-60 minute; Get supernatant, repeat above-mentioned extraction steps 2-4 time; Get supernatant, add the 5-15M LiCl solution of supernatant liquor 1/5-1/2 volume and mix ,-10 ℃--under 50 ℃ of conditions, place 10-20 hour, make RNA precipitation; 0-20 ℃, 5000-20000 g, centrifugal 5-60 minute, abandons supernatant, obtains total RNA precipitation of slightly putting forward;
Described RNA Extraction buffer is a kind of aqueous solution, the concentration that in this solution, the massfraction of CTAB is 2%, the massfraction of PVP40 is 2%-5%, Tris-Cl is that the concentration of 100 mM, EDTA is 25 mM at pH 8.0 at pH 8.0, the concentration of NaCl is 2 M at pH 8.0, the massfraction of β-ME is 2-5%, and wherein β-ME added in the used time;
(3) purifying of Kiwifruit RNA; Add DEPC treated water to dissolve total RNA obtained above, wherein DEPC treated water is 5:1-20:1 with the volume mass ratio of total RNA, the 1-10 M NaAc that adds again DEPC treated water 1/30-1/3 volume, the ethanol of the 90%-98% of 1-10 volume; Be placed in-5 ℃--50 ℃, 1-5 hour, makes RNA precipitation; 0-20 ℃, 5000-20000 g, centrifugal 5-60 minute, abandons supernatant, by 50%-95% ethanol washing and precipitating, obtains RNA after air drying.
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| CN101486747A (en) * | 2009-02-20 | 2009-07-22 | 浙江省农业科学院 | Method for extracting plant DNA and RNA at the same time |
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| CN101486747A (en) * | 2009-02-20 | 2009-07-22 | 浙江省农业科学院 | Method for extracting plant DNA and RNA at the same time |
Non-Patent Citations (6)
| Title |
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| A Rapid and Effective Method for RNA Extraction from Different Tissues of Grapevin and Other Woody Plants;GIORGIO GAMBINO et al.;《Phytochemical Analysis》;20080710;第19卷;520-525 * |
| GIORGIO GAMBINO et al..A Rapid and Effective Method for RNA Extraction from Different Tissues of Grapevin and Other Woody Plants.《Phytochemical Analysis》.2008,第19卷520-525. |
| 一种高效提取猕猴桃DNA和RNA的方法;刘胜洪等;《生物技术通报》;20110930(第9期);第171页左栏第2段 * |
| 刘胜洪等.一种高效提取猕猴桃DNA和RNA的方法.《生物技术通报》.2011,(第9期),第171页左栏第2段. |
| 提取猕猴桃果实总RNA的新方法;梅晓宏等;《中国食品学报》;20070630;第7卷(第3期);第55页右栏第2段 * |
| 梅晓宏等.提取猕猴桃果实总RNA的新方法.《中国食品学报》.2007,第7卷(第3期),第55页右栏第2段. |
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