CN110760508A - Method for extracting total RNA of wheat - Google Patents
Method for extracting total RNA of wheat Download PDFInfo
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- CN110760508A CN110760508A CN201910396980.4A CN201910396980A CN110760508A CN 110760508 A CN110760508 A CN 110760508A CN 201910396980 A CN201910396980 A CN 201910396980A CN 110760508 A CN110760508 A CN 110760508A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The invention discloses a method for extracting total RNA of wheat, which comprises the following steps: step 1: taking fresh or wheat tissue stored in a refrigerator at-70 ℃, and adding liquid nitrogen in the grinding process to keep the powder in a frozen state; obtaining wheat flour; step 2: transferring the wheat powder into a 1-5ml centrifuge tube A; placing the centrifugal tube A on ice to obtain a wheat sample mixed solution A; and step 3: adding an isometric chloroform solution into the wheat sample mixed solution obtained in the step 2, and uniformly mixing for 2-3 times; obtaining a wheat sample mixed solution B; and 5: adding isopropanol with the volume 0.25 times that of the centrifuge tube B and RNA lysate with the volume 0.25 times that of the centrifuge tube B into the centrifuge tube B, and filling the centrifuge tube B with the clear liquid obtained in the step 4; mixing, and standing on ice for 30 min; and 7: adding appropriate amount of 0.1% DEPC treated water to dissolve, and collecting dissolved substance as RNA of wheat tissue. The invention eliminates the use of sodium acetate and acidic phenol, and the experimental result shows that the yield and the quality of RNA can not be reduced.
Description
Technical Field
The invention relates to the technical field of plant biology, in particular to a method for extracting total RNA of wheat.
Background
RNA with good integrity and high purity is the premise and the basis for molecular biology research such as wheat RT-PCR, Northern blot analysis, cDNA library construction and the like. Wheat contains various histiocytes rich in secondary substances such as polysaccharide, lipid, polyphenol, pigment and the like, which are difficult to remove in the RNA extraction process. The existence of polysaccharide and lipid can not only reduce the solubility of RNA, but also inhibit the activity of a plurality of tool enzymes; secondary biomasses such as polyphenol, pigment and the like are easily oxidized in the extraction process to cause RNA browning, and the RNA is influenced to be used for further molecular operation, so whether the interfering substances can be removed as much as possible in the extraction process is the key point for obtaining high-quality wheat total RNA.
Wheat contains phenolic substances which are released during the homogenization of the material, which, after oxidation, turn brown and deepen with increasing oxidation. The oxidized phenolic compound can be stably combined with RNA, thereby influencing the separation and purification of the RNA.
Contamination with polysaccharides is another problem often encountered in extracting wheat RNA. Wheat tissue is often rich in polysaccharides, many of which have physicochemical properties similar to those of RNA and are therefore difficult to separate. RNA is removed while polysaccharide is removed, so that the yield of RNA is reduced; in addition, gel-like precipitation of polysaccharide is also generated when RNA is precipitated, and the RNA precipitate containing polysaccharide is difficult to dissolve in water or generates a viscous solution after dissolution. Since polysaccharides can inhibit the activity of many enzymes, RNA samples contaminated with polysaccharides cannot be used for further molecular biology studies.
Protein is yet another important factor for contaminating RNA samples. Since RNase and polyphenol oxidase also belong to proteins, it is necessary to remove protein impurities efficiently in order to obtain intact, high-quality RNA.
Ubiquitous exogenous and endogenous RNases are the most troublesome problem in RNA extraction. These RNases have the potential to degrade RNA preparations.
The existing methods for extracting the total RNA of the wheat have many methods, but the extraction effect of the methods in actual operation is not ideal, mainly because the methods can not effectively eliminate the influence of polysaccharide, lipid and the like, impurities often remain on an interface during extraction, the requirement can be met only by repeatedly extracting, the required time is long, and the integrity and the yield of the total RNA are also influenced. Meanwhile, the experimental methods have the disadvantages of high cost of used medicines, complicated procedures, long time, low quality of extracted RNA and influence on later use.
Disclosure of Invention
The invention aims to provide a method for extracting total RNA of wheat, which solves the problems that the existing methods for extracting the total RNA of the wheat are many, but the extraction effect of the methods in actual operation is not ideal.
In order to achieve the purpose, the invention provides the following technical scheme: a method for extracting wheat total RNA comprises the following steps:
step 1: taking fresh or wheat tissue preserved in a refrigerator at-70 ℃, adding liquid nitrogen into a mortar, and grinding the wheat tissue into powder by using a pestle; adding liquid nitrogen in the grinding process to keep the powder in a frozen state; obtaining wheat flour;
step 2: transferring the wheat powder into a 1.5ml centrifuge tube A, adding 1ml of ice-cold lysis solution into every 50-100 mg of wheat powder, and uniformly oscillating; placing the centrifugal tube A on ice to obtain a wheat sample mixed solution A;
and step 3: adding an isometric chloroform solution into the wheat sample mixed solution obtained in the step 2, violently shaking and uniformly mixing the sample, carrying out ice bath for 15min, and uniformly mixing for 2-3 times; obtaining a wheat sample mixed solution B;
and 4, step 4: centrifuging at 4 deg.C at 12000r/min for 15min to separate the wheat sample mixed solution B into two phases, and collecting the upper water phase; obtaining clear liquid;
and 5: adding isopropanol with the volume 0.25 times that of the centrifuge tube B and RNA lysate with the volume 0.25 times that of the centrifuge tube B into the centrifuge tube B, and filling the centrifuge tube B with the clear liquid obtained in the step 4; mixing, and standing on ice for 30 min;
step 6: centrifuging at 4 deg.C at 12000r/min for 15min, removing the upper layer liquid, and collecting the precipitate as RNA precipitation solution; washing the precipitate with 75% ethanol for 2 times; the centrifuge tube B with the tube cover opened is slightly blown dry on an ultra-clean bench so that the ethanol is evaporated (care is taken not to dry out, otherwise, the ethanol is difficult to dissolve);
and 7: adding appropriate amount of 0.1% DEPC treated water to dissolve, and collecting dissolved substance as RNA of wheat tissue.
Preferably, the RNA lysate prepared in step 5 is prepared from 47.28g of guanidine thiocyanate, 1.2352g of sodium citrate, 0.83g of sodium lauryl sarcosine, 1.5g of PVP (polyvinylpyrrolidone) and 100ul of β -mercaptoethanol, and DEPC-treated water is added to 100 ml.
Preferably, the chloroform solution in step 3 is a mixture of 24 parts by volume of chloroform and 1 part by volume of isoamyl alcohol.
Preferably, the RNA precipitation solution in the step 6 is 1.2mol/L NaCl and 0.8mol/L sodium citrate.
Preferably, the 75% ethanol in step 6 is formulated with 0.1% DEPC treated water.
Preferably, the DEPC treated water: 0.1mol of DEPC (diethylpyrocarbonate) was added to 100ml of double distilled water, treated overnight at 37 ℃ and then inactivated by autoclaving.
Preferably, the centrifuge tube a and the centrifuge tube B are soaked in 0.1% DEPC water overnight and then autoclaved.
Preferably, the pestle and the mortar are treated by DEPC water and precooled; preparing the wheat total RNA.
Compared with the prior art, the invention has the following beneficial effects:
the method is based on the traditional guanidinium isothiocyanate-chloroform method, adds some auxiliary agents and simplifies some steps to extract the cotton total RNA.
(1) The invention eliminates the use of sodium acetate and acidic phenol, and experimental results show that the yield and quality of RNA are not reduced, and the pollution of exogenous RNase possibly brought by the reagent is reduced, and the extraction time is shortened.
(2) When the RNA is precipitated, isopropanol with the volume of 1/4 lysate and RNA precipitation solution with the volume of 1/4 lysate are added, so that the pollution level of polysaccharide and proteoglycan can be greatly reduced.
(3) The addition of PVP can effectively prevent the oxidation of the phenolic substances, thereby preventing the irreversible combination of the oxides of the phenolic substances and RNA and effectively avoiding the interference of the phenolic substances.
(4) The steps are simple and the time is short. The less chance that various harmful factors will damage nucleic acid, the less chance of RNA degradation and RNA loss while maintaining RNA yield and quality. The extracted RNA has high quality and no degradation, and improves the research of wheat in molecular biology.
(5) The required medicines are few in types and are all conventional medicines, the price is low, and the cost is low
(6) The used material is not limited, and fresh material can be used, and the material preserved at-70 deg.C can be used, and wheat material grown in field can also be used.
(7) The repeatability is good, and reference is provided for RNA extraction of other plants.
Drawings
FIG. 1 shows total RNA extracted from wheat of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, a method for extracting total RNA from wheat includes the following steps:
step 1: taking fresh or wheat tissue preserved in a refrigerator at-70 ℃, adding liquid nitrogen into a mortar, and grinding the wheat tissue into powder by using a pestle; adding liquid nitrogen in the grinding process to keep the powder in a frozen state; obtaining wheat flour;
step 2: transferring the wheat powder into a 1.5ml centrifuge tube A, adding 1ml of ice-cold lysis solution into every 50-100 mg of wheat powder, and uniformly oscillating; placing the centrifugal tube A on ice to obtain a wheat sample mixed solution A;
and step 3: adding an isometric chloroform solution into the wheat sample mixed solution obtained in the step 2, violently shaking and uniformly mixing the sample, carrying out ice bath for 15min, and uniformly mixing for 2-3 times; obtaining a wheat sample mixed solution B;
and 4, step 4: centrifuging at 4 deg.C at 12000r/min for 15min to separate the wheat sample mixed solution B into two phases, and collecting the upper water phase; obtaining clear liquid;
and 5: adding isopropanol with the volume 0.25 times that of the centrifuge tube B and RNA lysate with the volume 0.25 times that of the centrifuge tube B into the centrifuge tube B, and filling the centrifuge tube B with the clear liquid obtained in the step 4; mixing, and standing on ice for 30 min;
step 6: centrifuging at 4 deg.C at 12000r/min for 15min, removing the upper layer liquid, and collecting the precipitate as RNA precipitation solution; washing the precipitate with 75% ethanol for 2 times; the centrifuge tube B with the tube lid open was blown slightly dry on an ultra clean bench to allow for ethanol evaporation (care not to dry out, otherwise difficult to dissolve);
and 7: adding a proper amount of 0.1 percent DEPC treated water for dissolving, and collecting a dissolved substance, namely the RNA of the wheat tissue;
furthermore, the proportion of the RNA lysate in the step 5 is that 47.28g of guanidine thiocyanate, 1.2352g of sodium citrate, 0.83g of sodium lauryl sarcosine, 1.5g of PVP (polyvinylpyrrolidone) and 100ul of β -mercaptoethanol are used, and DEPC treated water is added to reach 100 ml.
Further, the chloroform solution in step 3 is a mixture of 24 parts by volume of chloroform and 1 part by volume of isoamyl alcohol.
Further, the RNA precipitation solution in the step 6 is 1.2mol/L NaCl and 0.8mol/L sodium citrate.
Further, the 75% ethanol in step 6 was formulated with 0.1% DEPC treated water.
Further, the DEPC treated water: 0.1mol of DEPC (diethylpyrocarbonate) was added to 100ml of double distilled water, treated overnight at 37 ℃ and then inactivated by autoclaving.
Further, both centrifuge tube a and centrifuge tube B were soaked overnight in 0.1% DEPC water and then autoclaved.
Further, the pestle and the mortar are treated by DEPC water and precooled; preparing the wheat total RNA.
The wheat total RNA prepared by the method is detected according to the following modes:
and (3) determining the RNA content by an ethidium bromide fluorescence method: preparing 1% agarose gel, taking 1ul RNA sample for electrophoresis, staining for 15min by ethidium bromide after electrophoresis, irradiating under a short-wavelength ultraviolet lamp, and visually observing the quality and content of RNA.
Nucleic acid protein detection is performed according to the method: and (3) measuring the absorbance values of the purified RNA solution under the illumination of 260nm and 280nm wavelengths by using a nucleic acid protein detector to determine the concentration, and detecting the purity and the content of the RNA.
The result of detecting and extracting genome RNA of multiple groups of wheat total RNA according to the method is that 28S and 18S 2 bands can be clearly observed after staining on 1% agarose gel, and the OD260/OD280 value of the RNA sample is determined to be more than 1.8, which shows that the RNA is not polluted by protein and phenol, and is high in purity, as shown in figure 1, the total RNA extracted from the tender leaves of wheat is extracted.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A method for extracting total RNA of wheat is characterized by comprising the following steps:
step 1: taking fresh or wheat tissue preserved in a refrigerator at-70 ℃, adding liquid nitrogen into a mortar, and grinding the wheat tissue into powder by using a pestle; adding liquid nitrogen in the grinding process to keep the powder in a frozen state; obtaining wheat flour;
step 2: transferring the wheat powder into a 1.5ml centrifuge tube A, adding 1ml of ice-cold lysis solution into every 50-100 mg of wheat powder, and uniformly oscillating; placing the centrifugal tube A on ice to obtain a wheat sample mixed solution A;
and step 3: adding an isometric chloroform solution into the wheat sample mixed solution A obtained in the step 2, violently shaking and uniformly mixing the sample, carrying out ice bath for 15min, and uniformly mixing for 2-3 times; obtaining a wheat sample mixed solution B;
and 4, step 4: centrifuging at 4 deg.C at 12000r/min for 15min to separate the wheat sample mixed solution B into two phases, and collecting the upper water phase; obtaining clear liquid;
and 5: adding isopropanol with the volume 0.25 times that of the centrifuge tube B and RNA lysate with the volume 0.25 times that of the centrifuge tube B into the centrifuge tube B, and filling the centrifuge tube B with the clear liquid obtained in the step 4; mixing, and standing on ice for 30 min;
step 6: centrifuging at 4 deg.C at 12000r/min for 15min, removing the upper layer liquid, and collecting the precipitate as RNA precipitation solution; washing the precipitate with 75% ethanol for 2 times; the centrifuge tube B with the tube cover opened is slightly blown dry on an ultra-clean bench so that the ethanol is evaporated (care is taken not to dry out, otherwise, the ethanol is difficult to dissolve);
and 7: adding appropriate amount of 0.1% DEPC treated water to dissolve, and collecting dissolved substance as RNA of wheat tissue.
2. The method for extracting total RNA from wheat as claimed in claim 1, wherein the RNA lysate obtained in step 5 is prepared from 47.28g of guanidinium thiocyanate, 1.2352g of sodium citrate, 0.83g of sarcosyl, 1.5g of PVP (polyvinylpyrrolidone) and 100ul of β -mercaptoethanol, and DEPC-treated water to 100 ml.
3. The method for extracting total RNA from wheat as claimed in claim 1, wherein the chloroform solution in step 3 is a mixture of 24 parts by volume of chloroform and 1 part by volume of isoamyl alcohol.
4. The method for extracting total RNA from wheat as claimed in claim 1, wherein the RNA precipitation solution in step 6 is 1.2mol/L NaCl and 0.8mol/L sodium citrate.
5. The method for extracting total RNA from wheat as claimed in claim 1, wherein the 75% ethanol in step 6 is prepared with 0.1% DEPC treated water.
6. The method for extracting wheat total RNA as claimed in claim 5, wherein the DEPC treated water; 0.1mol of DEPC (diethylpyrocarbonate) was added to 100ml of double distilled water, treated overnight at 37 ℃ and then inactivated by autoclaving.
7. The method for extracting total RNA from wheat as claimed in claim 1, wherein the centrifuge tube A and the centrifuge tube B are immersed in 0.1% DEPC water overnight and then autoclaved.
8. The method for extracting the total RNA of wheat according to claim 1, wherein the pestle and the mortar are treated with DEPC water and pre-cooled; preparing the wheat total RNA.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1900278A (en) * | 2006-06-30 | 2007-01-24 | 西北农林科技大学 | Extracting method for wheat seed total RNA |
| 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 |
| CN102453709A (en) * | 2010-10-19 | 2012-05-16 | 于婷婷 | Extraction process for plant RNA |
| CN102776174A (en) * | 2012-08-21 | 2012-11-14 | 昆明理工大学 | Method for simultaneously extracting DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) from lily tissue |
| CN103740700A (en) * | 2013-12-31 | 2014-04-23 | 华南师范大学 | Extracting method for total RNA (Ribonucleic Acid) of galangal |
-
2019
- 2019-05-08 CN CN201910396980.4A patent/CN110760508A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1900278A (en) * | 2006-06-30 | 2007-01-24 | 西北农林科技大学 | Extracting method for wheat seed total RNA |
| 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 |
| CN102453709A (en) * | 2010-10-19 | 2012-05-16 | 于婷婷 | Extraction process for plant RNA |
| CN102776174A (en) * | 2012-08-21 | 2012-11-14 | 昆明理工大学 | Method for simultaneously extracting DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) from lily tissue |
| CN103740700A (en) * | 2013-12-31 | 2014-04-23 | 华南师范大学 | Extracting method for total RNA (Ribonucleic Acid) of galangal |
Non-Patent Citations (6)
| Title |
|---|
| 南海波: "小麦总RNA的提取", 《渤海大学学报（自然科学版）》 * |
| 崔素萍: "一种快速提取小麦叶片总RNA的方法", 《西北植物学报》 * |
| 李浩: "高质量的小麦种子总RNA的快速提取方法", 《分子植物育种》 * |
| 杜何为: "小麦总RNA的小量快速提取", 《湖北农业科学》 * |
| 杜何为: "水稻、玉米、小麦成熟种子总RNA的快速提取", 《分子植物育种》 * |
| 龚莉桂: "小麦总RNA的快速提取", 《激光生物学报》 * |
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Application publication date: 20200207 |