CN102690806A - Method for simply and rapidly extracting total RNA from switchgrass tissue - Google Patents
Method for simply and rapidly extracting total RNA from switchgrass tissue Download PDFInfo
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Abstract
The invention provides a method for simply and rapidly extracting total RNA from switchgrass tissue, and pertains to the field of biotechnology. The concrete steps of the method comprises: a first step of preparing experimental medicines and extracting biological samples from switchgrass leaves or stems; a second step of releasing nucleic acid from the biological samples by using chemical reagents; a third step of extracting free RNA by using chloroform and precipitating the free RNA by using isopropanol to obtain the total RNA; and a fourth step of identifying the total RNA by using an ultraviolet spectrophotometer and an agarose gel electrophoresis detection method separately. The electrophoresis and data results show that the obtained RNA of plant tissues has good integrity, high purity and high yield, and can meet requirements in molecular biology research and application. Meanwhile, the method has simple experimental process, easy operation, good security, accurate results, good availability, and good repeatability.
Description
Technical field
The invention belongs to biological technical field, relate to gene clone, the gene function analysis equimolecular biological experiment of plant, be specifically related to the method for a kind of simple, total RNA of rapid extraction plant tissue, be applicable to the extraction of the total RNA of plant tissue.
Background technology
Along with the development of modern molecular biology and to the infiltration of each subject, increasing molecular level technology is applied to Study on plants.As adopt the resistance of transgenic technology enhancement of plant, and improve photosynthetic efficiency, increase biological yield etc.The basic experiment technology that the total RNA extractive technique of plant is a molecular biology of plants also is the prerequisite of carrying out the research of molecular biology of plants aspect, and is significant for functional genomics and genetics research.Utilize to separate that the purity that obtains is high, the total RNA of high quality of good in integrity, just can be used for Northern hybridization, mRNA purifying, construction cDNA library are through the RT-PCR isolated genes and carry out aleuroplast and translate the operation of equimolecular biological experiment outward.Because all there are a large amount of RNA enzymes in nature and laboratory, so the extraction product of RNA very easily degrades, and brings bigger difficulty to experiment.Therefore how rapid extraction RNA and prevent the degraded of RNA and make it to store the long time is the difficult problem that the molecular biology research personnel will solve always.Although the extraction of RNA has become very proven technique, in practical study, often be difficult to accomplish to obtain smoothly the measured RNA of matter.This is attributed to several factors, as material preserve unreasonable, vessel are unholiness, operation is not tight etc., cause the degraded of RNA.
The process for extracting of relevant plant RNA is more; Existing method mainly contains guanidine isothiocyanate method, SDS/ phenol method, CTAB-LiCL method etc.; These methods have been widely used in the extraction of the total RNA of many plant tissues; But there is in some plant tissues inclusion very complicated, especially, is unfavorable for the separation and purification of RNA being rich in polysaccharide, polyphenol, tannin and some still under the situation such as doubtful secondary metabolite.Shortcomings such as all there is certain defective in these methods when using, long like extraction time, and it is harsh to extract required condition, and extraction yield is low, and total RNA integrity of extraction is relatively poor.Yet the quality of RNA quality is determining to test result's confidence level height.Therefore obtain a kind of simply, the plant tissue method for extracting total RNA is significant fast.
Switchgrass (English name Switchgrass, latin name
Panicum virgatumlinn), claim " energy grass " again, because of its cultivation technique simple; Wide adaptability, the ability of checking winds and fixing drifting sand is strong, impoverishment tolerant; Biological yield is high, is confirmed as in the world and produces the first-selected bioenergy plant of dyestuff alcoholic acid, and dropped into great amount of manpower and material resources and researched and developed.Wherein, adopt genetic engineering technique to increase biological yield, reduce content of lignin and become one of present research focus with aspects such as improving alcohol yied.And the extraction of high quality high purity RNA is one of chief component of biotechnology, in the foundation of genetic conversion system, has crucial meaning.Switchgrass is more because of cellulose and other impurity, make its difficult grinding thoroughly in the process of lapping that extracts the RNA initial stage, and it is long to expend time in, and influences switchgrass RNA extraction efficiency.
A kind of simple, the total RNA method of rapid extraction plant tissue that the present invention proposes is the important prerequisite that plant carries out transgenic technology.The objective of the invention is to provide a kind of and can make the method for extracting total RNA that time is shorter, simple to operate, with low cost, extraction efficiency is higher that extracts the total RNA of plant, realize by following mode.At first in process of lapping, add the wolfram varbide grinding bead, make milling time shorten, and grinding effect is better.Secondly total RNA extracting solution contains highly efficient depressor, washing agent, the pH regulator agent of RNA enzyme.Highly efficient depressor has guanidinium isothiocyanate, water-saturated phenol, and washing agent is a sarcosyl, and the pH regulator agent is the NaAc damping fluid.This RNA extracting solution cost is lower, and easy being easy to get.Method for extracting total RNA of the present invention comprises sample grinding, nucleic acid release, chloroform extracting, isopropanol precipitating, air-dry five steps of cleaning; Leaching process is simple; Running time short (30-40min), economical and effective, extraction yield is high; Total RNA good in integrity of extracting is applicable to the molecular biology of plants operation.
Summary of the invention
The purpose of this invention is to provide the method for a kind of simple, total RNA of rapid extraction plant tissue, simple to operate, extraction time is short; Good reproducibility, suitability is strong, can obtain the RNA of high quality, high density; For further molecular biology research is laid a good foundation, accurate and effective as a result.
The method of a kind of simple, rapid extraction switchgrass total tissue RNA provided by the present invention, carry out according to following step:
(1) sample grinds: get fresh switchgrass blade of about 0.5 ~ 1g or stem material in mortar; Add 3 ~ 5 of 3mm wolfram varbide grinding bead; Grind rapidly after adding liquid nitrogen; Repeat several until claying into power, milling time is controlled at 1min, the powder after grinding is added in the 1.5mL EP pipe of precooling;
(2) nucleic acid discharges: add the RNA extracting solution that 1mL prepares in the centrifuge tube in step (1), fully vibration mixes it, parallel placement 8-10min;
(3) the centrifugal 5 ~ 8min of chloroform extracting: 12000 ~ 14000r/min gets supernatant 600 ~ 800 microlitres and changes in the new 1.5mL EP pipe, adds 250 ~ 300 microlitre chloroforms of precooling, and vortex 15s on vortice makes its abundant mixing;
(4) the centrifugal 8 ~ 10min of isopropanol precipitating: 12000 ~ 14000r/min gets upper strata water 200 ~ 300 microlitres
Change in the new 1.5mL EP pipe, add 200 ~ 300 microlitres
Virahol turns upside down for several times;
(5) clean: the centrifugal 5 ~ 8min of 12000 ~ 14000r/min, outwell supernatant, add volume(tric)fraction and be 70-80% washing with alcohol deposition;
(6) taking precipitate dries up about 8-10min in super clean bench, and the adding volume(tric)fraction is 0.1 ‰ ~ 0.1% diethylpyrocarbonate (DEPC water) dissolving, gets total RNA.
Wherein the every 100ml of RNA extracting solution described in the step (2) contains the water-saturated phenol of 20 ~ 30ml, the glycerine of 4 ~ 5ml, the guanidinium isothiocyanate of 0.1 ~ 0.2mol, the NaAc damping fluid of the pH=4.0 of 0.01 ~ 0.02mol, the sarcosyl of 1 ~ 1.5ml.
The invention has the advantages that:
1, extraction time lacks, and whole extraction operating process is accomplished in 40min.
2, extract the total RNA good in integrity of plant, the absorbance ratio of plant leaf total rna solution under uv-absorbing wavelength 260nm and 280nm approaches 2,5s wherein, and 18s, the 28s band is clear, and the brightness ratio of 28s band and 18s band is near 2.
3, extraction efficiency is high, and the RNA amount is big, can be used for the library, Northern blot etc.
4, the cost of RNA extracting solution is lower, and efficient is higher.
Description of drawings
Fig. 1 is that agarose gel electrophoresis detects the switchgrass total tissue RNA, and with the take pictures design sketch of the experimental result that write down of gel imaging system, wherein 1,2 material therefors are the mixture of switchgrass blade and stem behind the electrophoresis.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.
Embodiment 1
The preparation of A, experimental drug: RNA extracting solution, volume(tric)fraction are 0.1 ‰ DEPC water, and volume(tric)fraction is 70% ethanol (preparation of 0.1 ‰ DEPC water).
The processing of B, experimental article: it is after 0.1 ‰ DEPC water loggings bubble spends the night that vessel use volume(tric)fraction, 120 ℃ of sterilization 80min, and it is 2% hydrogen peroxide dipping 30min that electrophoresis chamber and application of sample comb use volume(tric)fraction.
C, draw materials: switchgrass blade and stem
The extraction of D, total RNA:
(1) sample grinds: get fresh switchgrass blade of about 0.5g or stem material in mortar; Add 3 of 3mm wolfram varbide grinding bead, grind rapidly behind the adding liquid nitrogen, repeat for several times until claying into power; Milling time is controlled at 1min, the powder after grinding is added in the 1.5mL EP pipe of precooling;
(2) nucleic acid discharges: add the RNA extracting solution that 1mL prepares in the centrifuge tube in step (1), fully vibration mixes it, parallel placement 8min;
(3) the centrifugal 5min of chloroform extracting: 12000r/min gets supernatant 600 microlitres
Change in the new 1.5mL EP pipe, add 250 microlitres of precooling
Chloroform, vortex 15s on vortice makes its abundant mixing;
(4) the centrifugal 8min of isopropanol precipitating: 12000 ~ 14000r/min gets upper strata water 200 microlitres
Change in the new 1.5mL EP pipe, add 200 microlitres
Virahol turns upside down for several times;
(5) clean: the centrifugal 5min of 12000 ~ 14000r/min, outwell supernatant, adding volume(tric)fraction is 70% washing with alcohol deposition;
(6) taking precipitate dries up about 8min in super clean bench, and the adding volume(tric)fraction is 0.1 ‰ diethylpyrocarbonate (DEPC water) dissolving, gets total RNA.
E, RNA sample yield, purity and integrity are identified:
(1) agarose gel electrophoresis detects: the integrity that detects RNA with 1% agarose gel electrophoresis.Get 1 microlitre
RNA solution, 4 microlitres
DEPC water, 1 microlitre
Appearance on 6 * loading buffer.The electrophoresis detection condition, voltage 120V, electric current 80mA, constant voltage, damping fluid is that volume(tric)fraction is 1% TAE.The electrophoresis picture of the total RNA of switchgrass leaf tissue is as shown in Figure 1, visible 5s, and 18s and 28s band are clear.
(2) uv absorption spectrum detects: the A that measures institute's extracting RNA sample with ultraviolet spectrophotometer
260And A
280Value.(1) is the detected result of switchgrass total tissue RNA: OD among the figure
260=0.033, OD
280=0.064, OD
260/ OD
280=2.1, OD
260/ OD
230=2.3, yield=256ng/ microlitre
。OD
260/ OD
280Ratio approach 2.0 more, explain that the RNA purity of being extracted is high more.Work as OD
260/ OD
230>2.0 the time, explaining in the RNA sample that is extracted does not have saline pollution.The RNA yield that method of use is extracted is higher, and purity meets the requirements.
Embodiment 2
The preparation of A, experimental drug: RNA extracting solution, volume(tric)fraction are 0.1%DEPC water, and volume(tric)fraction is 80% ethanol (preparation of 0.1%DEPC water).
The processing of B, experimental article: after vessel use volume(tric)fraction to spend the night as 0.1%DEPC water logging bubble, 120 ℃ of sterilization 100min, it is 3% hydrogen peroxide dipping 50min that electrophoresis chamber and application of sample comb use volume(tric)fraction.
C, draw materials: switchgrass blade and stem
The extraction of D, total RNA:
(1) sample grinds: get fresh switchgrass blade of about 1g or stem material in mortar; Add 5 of 3mm wolfram varbide grinding bead, grind rapidly behind the adding liquid nitrogen, repeat for several times until claying into power; Milling time is controlled at 1min, the powder after grinding is added in the 1.5mL EP pipe of precooling;
(2) nucleic acid discharges: add the RNA extracting solution that 1mL prepares in the centrifuge tube in step (1), fully vibration mixes it, parallel placement 10min;
(3) the centrifugal 8min of chloroform extracting: 14000r/min gets supernatant 800 microlitres
Change in the new 1.5mL EP pipe, add 300 microlitres of precooling
Chloroform, vortex 15s on vortice makes its abundant mixing;
(4) the centrifugal 10min of isopropanol precipitating: 14000r/min gets upper strata water 300 microlitres
Change in the new 1.5mL EP pipe, add 300 microlitres
Virahol turns upside down for several times;
(5) clean: the centrifugal 8min of 14000r/min, outwell supernatant, adding volume(tric)fraction is 80% washing with alcohol deposition;
(6) taking precipitate dries up about 10min in super clean bench, and the adding volume(tric)fraction is 0.1% diethylpyrocarbonate (DEPC water) dissolving, gets total RNA.
E, RNA sample yield, purity and integrity are identified: with embodiment 1.
?
Claims (2)
1. the method for simple, a rapid extraction switchgrass total tissue RNA is characterized in that carrying out according to following step:
(1) sample grinds: get fresh switchgrass blade of about 0.5 ~ 1g or stem material in mortar; Add 3 ~ 5 of 3mm wolfram varbide grinding bead; Grind rapidly after adding liquid nitrogen; Repeat several until claying into power, milling time is controlled at 1min, the powder after grinding is added in the 1.5mL EP pipe of precooling;
(2) nucleic acid discharges: add the RNA extracting solution that 1mL prepares in the centrifuge tube in step (1), fully vibration mixes it, parallel placement 8-10min; Microlitre
(3) the centrifugal 5 ~ 8min of chloroform extracting: 12000 ~ 14000r/min gets supernatant 600 ~ 800 microlitres
Change in the new 1.5mL EP pipe, add 250 ~ 300 microlitre chloroforms of precooling, vortex 15s on vortice makes its abundant mixing;
(4) the centrifugal 8 ~ 10min of isopropanol precipitating: 12000 ~ 14000r/min gets upper strata water 200 ~ 300 microlitres and changes in the new 1.5mL EP pipe, adds 200 ~ 300 microlitre Virahols, turns upside down for several times;
(5) clean: the centrifugal 5 ~ 8min of 12000 ~ 14000r/min, outwell supernatant, add volume(tric)fraction and be 70-80% washing with alcohol deposition;
(6) taking precipitate dries up about 8-10min in super clean bench, and the adding volume(tric)fraction is 0.1 ‰ ~ 0.1% diethylpyrocarbonate dissolving, gets total RNA.
2. the method for a kind of simple, rapid extraction switchgrass total tissue RNA according to claim 1; It is characterized in that the every 100ml of RNA extracting solution described in the step (2) wherein contains the water-saturated phenol of 20 ~ 30ml; The glycerine of 4 ~ 5ml; 0.1 the guanidinium isothiocyanate of ~ 0.2mol, the NaAc damping fluid of the pH=4.0 of 0.01 ~ 0.02mol, the sarcosyl of 1 ~ 1.5ml.
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| CN102899318A (en) * | 2012-10-31 | 2013-01-30 | 四川省林业科学研究院 | Method for extracting nanmu RNA (Ribonucleic Acid) |
| CN103074330A (en) * | 2013-02-26 | 2013-05-01 | 昆明理工大学 | Method for efficiently extracting solanaceae seed RNA (Ribonucleic Acid) |
| CN106282169A (en) * | 2016-11-01 | 2017-01-04 | 中国水产科学研究院淡水渔业研究中心 | A kind of rapid batch extracts the method for fish tissues total serum IgE |
| CN108410865A (en) * | 2018-05-31 | 2018-08-17 | 山东省花生研究所 | One method for cultivating peanut DNA rapid extractions |
| CN112280776A (en) * | 2020-11-05 | 2021-01-29 | 广西民族师范学院 | Wild Ganoderma applanatum RNA extraction method |
| CN112410402A (en) * | 2020-11-12 | 2021-02-26 | 麦凯(上海)生物科技有限公司 | Free RNA precipitation aid |
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| CN106282169A (en) * | 2016-11-01 | 2017-01-04 | 中国水产科学研究院淡水渔业研究中心 | A kind of rapid batch extracts the method for fish tissues total serum IgE |
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| CN112280776A (en) * | 2020-11-05 | 2021-01-29 | 广西民族师范学院 | Wild Ganoderma applanatum RNA extraction method |
| CN112410402A (en) * | 2020-11-12 | 2021-02-26 | 麦凯(上海)生物科技有限公司 | Free RNA precipitation aid |
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Application publication date: 20120926 |