CN110592075A - Method for rapidly and efficiently extracting RNA (ribonucleic acid) of fermented grains - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to a method for quickly and efficiently extracting RNA from fermented grains. The method comprises the following steps: pre-treating a fermented grain sample, extracting total RNA of the fermented grain sample and removing DNA in the total RNA of the sample. The method has the beneficial effects that: firstly, extracting the total RNA of the microorganism by using a thermal phenol method in the process of brewing the white spirit; the pretreatment step is beneficial to comprehensively obtaining microorganisms in the fermented grains and the distiller's yeast; the buffer solution containing sucrose is used for extraction, which is beneficial to obtaining RNA with higher quality; 1/10 volumes of 3mol/L sodium acetate pH 5.2 were added prior to extraction with acid phenol to aid in RNA and protein fractionation. The method disclosed by the invention is low in cost, convenient to operate, free of environmental pollution and small in harm to human bodies, and can be used for quickly and efficiently extracting RNA from fermented grains, which is of great importance for the research on microbial total RNA extraction in the process of brewing white spirit.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for quickly and efficiently extracting RNA from fermented grains.
Background
The quantity of microorganisms in the white spirit brewing is huge, the fermentation components are extremely complex, more than thousands of chemical substances are detected at present, and most of the substances are generated by the metabolism of the microorganisms in the raw materials or the fermentation process. Fermented grains are an important source for synthesizing flavor substances in the fermentation process, so the fermented grains are carriers of new flavor substances. In recent years, various domestic white spirit enterprises or scientific research institutes of colleges and universities continuously explore flavor substances in white spirit, and hopefully, new functional factors can be found, and the problems of complex technical operation, large workload, high equipment requirement, high cost and the like exist in the new flavor substances found in the white spirit brewing process at present. Meanwhile, for the research and analysis of the determination method of the content of trace components in the white spirit, the quantitative analysis method of compounds in products such as Daqu, fermented grains and vinasse in the solid fermentation process is always a research hotspot.
The extraction of RNA is a basic technology of modern molecular biology, and how to obtain high-quality RNA becomes the key for people to research whether life science is successful or not by using molecular biology technology. At present, the main methods for extracting RNA include an acid phenol method, a cold phenol method, a CTAB method, an SDS method, a phenol method, a Trizol method and the like. However, the above method has unsatisfactory extraction effect, short extraction time, and high cost. In other RNA extraction method steps, even if acidic phenol extraction is used for removing protein and DNA, the effect is not ideal, and the quality of extracted total RNA is not high. Therefore, the development of a method which has the advantages of low cost, convenient operation, no pollution to the environment, small harm to the human body and rapid and efficient extraction of RNA in fermented grains is urgently needed, and is very important for the research of the extraction of the total RNA of the microorganisms in the process of brewing the white spirit.
Disclosure of Invention
Based on the problems, the invention aims to provide a method for quickly and efficiently extracting RNA from fermented grains.
A method for rapidly and efficiently extracting RNA from fermented grains comprises the following steps:
the method comprises the following steps: sample pretreatment:
(1) weighing 1g of solid sample, and suspending with 10mL of sterilized and precooled 0.1mol/L PBS buffer solution;
(2) adding four steel balls with the diameter of 1mm into the sample, and carrying out vortex oscillation treatment for 5min until the thalli are completely dispersed;
(3) placing the sample after the oscillation treatment at 4 ℃, centrifuging at 300rpm for 5min, sucking the supernatant and the white middle layer, and placing the supernatant and the white middle layer in a sterilized 50mL centrifuge tube;
(4) washing the precipitate with 10mL precooled 0.1mol/L PBS buffer solution, centrifuging at 4 deg.C and 300rpm for 5min, collecting supernatant and white intermediate layer, repeating the process for 2 times, and mixing with the collected sample;
(5) centrifuging all supernatants at 4 deg.C and 12000 rpm for 3 min, discarding the supernatant, and collecting cell precipitate;
(6) washing the cell precipitate with 10mL precooled 0.1mol/L PBS buffer solution, centrifuging at 4 deg.C and 12000 rpm for 3 min, and collecting the precipitate, and repeating the process for 2 times;
(7) filtering the collected precipitate, and freeze-drying at-80 deg.C or directly extracting DNA or RNA;
step two: extraction of total RNA of the sample:
(1) fully grinding the pretreated sample in liquid nitrogen until the sample cells are completely broken by taking the liquid nitrogen submerging the sample as a standard;
(2) weighing 0.1g of ground sample, placing the ground sample in a 1.5mL RNase-free Eppendorf tube, adding 750 mu L of STE solution and 750 mu L of water-saturated phenol, and uniformly mixing by oscillation;
(3) heating the mixed sample in 65 deg.C water bath for 10min, and fully shaking and mixing for 1 time every 2 min;
(4) the heated sample was centrifuged at 12000 rpm at 4 ℃ for 5min, and then the supernatant was transferred to a new 1.5mL RNase-free Eppendorf tube;
(5) 1/10 volumes of a mixture of 3mol/L sodium acetate with pH 5.2 and 200. mu.L acid phenol, chloroform and isoamyl alcohol were added to the supernatant, wherein the acid phenol: chloroform: isoamyl alcohol = 25: 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL Eppendorf tube without RNase, and extracting until no intermediate protein layer exists;
(6) and adding 200 mu L of chloroform-isoamyl alcohol mixed solution into the supernatant, wherein the ratio of chloroform: isoamyl alcohol = 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL RNase-free Eppendorf tube, and extracting until no layering exists;
(7) adding isopropanol with volume 1 time of that of the supernatant, standing at room temperature for 10min, centrifuging at 12000 rpm at 4 ℃ for 10min, and discarding the supernatant;
(8) washing the precipitate with 75% ethanol for 2 times, air drying at room temperature, adding ddH2O20 ~ 30 mu L, fully dissolving the precipitate;
(9) detecting the quality of RNA through electrophoresis, and simultaneously determining the concentration of RNA through Nanodrop;
step three: removal of DNA from total RNA in the sample:
(1) taking 10 mu g of total RNA, adding 3 ~ 5U of DNase1 into 100 mu L of reaction system, and reacting for 30 minutes at 37 ℃;
(2) the reaction system was supplemented with 600. mu.L of ddH2And adding 200 mu L of mixed solution of acid phenol, chloroform and isoamylol, wherein the acid phenol: chloroform: isoamyl alcohol = 25: 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL Eppendorf tube without RNase, and extracting until no intermediate protein layer exists;
(3) and (3) mixing 200 mu L of chloroform and isoamylol in the supernatant, wherein the ratio of chloroform: isoamyl alcohol = 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL Eppendorf tube without RNase, and extracting until no phenol layer exists;
(4) adding 1/10 volume of 3mol/L sodium acetate with pH of 5.2 and 2 times volume of anhydrous ethanol into the supernatant, standing at-70 deg.C for more than 1 hr, centrifuging at 12000 rpm at 4 deg.C for 10min, and removing supernatant;
(5) washing the precipitate with 75% ethanol for 2 times, air drying at room temperature, adding ddH2O 10 μ L, and dissolving the precipitate;
(6) agarose electrophoresis detection, and Nanodrop determination of RNA quality.
Further, the sample is fermented grains of large batch or fermented grains of second batch in the white spirit fermentation process.
Further, the 0.1mol/L PBS buffer solution comprises a stock solution and a diluent, wherein the stock solution: 34.0g of potassium dihydrogen phosphate is weighed and dissolved in 500mL of distilled water, the pH value is adjusted by 175 mL of 1mol/L sodium hydroxide solution, and the solution is diluted to 1000mL by the distilled water and then stored in a refrigerator; diluting liquid: the stock solution (1.25 mL) was diluted to 1000mL with distilled water, and the solution was dispensed into suitable containers and autoclaved at 121 ℃ for 15 min, and the final pH of the PBS buffer was 7.2.
Further, the STE solution is: 0.3mol/L of sucrose; Tris-HCl 25mmol/L at pH 8.0; EDTA 25mmol/L at pH 8.0; and (5) sterilizing for later use.
Compared with the prior art, the method has the beneficial effects that:
firstly, extracting the total RNA of the microorganism by using a thermal phenol method in the process of brewing the white spirit; the pretreatment step is beneficial to comprehensively obtaining microorganisms in the fermented grains and the distiller's yeast; the buffer solution containing sucrose is used for extraction, which is beneficial to obtaining RNA with higher quality; 1/10 volumes of 3mol/L sodium acetate pH 5.2 were added prior to extraction with acid phenol to aid in RNA and protein fractionation. The method has the advantages of low cost, convenient operation, no pollution to the environment and small harm to human bodies, and can quickly and efficiently extract RNA in fermented grains, which is very important for the research of microbial total RNA extraction in the process of brewing white spirit.
Drawings
FIG. 1 is a graph of RNA quality detection by electrophoresis of a fermented grain sample.
Detailed Description
The technical solutions of the present invention are described in further detail below with reference to specific embodiments, which are intended to be merely illustrative of the present invention and should not be construed as limiting.
Example 1
The microbial community structure and the succession rule of the fermentation of the Niuban mountain Erguotou by the method are utilized. In the establishing process of the method, a pre-test is carried out in advance, according to the fermentation process of the Niubashan mountain Erguotou, the sampling positions are the central positions and the edge positions of the upper part, the middle part and the lower part of a ground cylinder, 300g of the large-batch fermented grains are taken at 0, 3, 5, 7, 9, 13, 19, 23 and 28 th hour, the sampling position is the position where the center of the fermentation ground cylinder is 50cm away from the cylinder bottom, the large-batch fermented grains are placed in an aseptic sealed bag to be uniformly mixed, and the mixture is sent to a laboratory for later use within 10 min.
A method for rapidly and efficiently extracting RNA from fermented grains comprises the following steps:
the method comprises the following steps: pre-treating a large-batch fermented grain sample:
(1) weighing 1g of a large-batch fermented grain sample, and suspending the large-batch fermented grain sample by using 10mL of sterilized and precooled 0.1mol/L PBS buffer solution;
(2) adding four steel balls with the diameter of 1mm into the large-batch fermented grains sample, and carrying out vortex oscillation treatment for 5min until the thalli are completely dispersed;
(3) placing the shaking treated fermented grains sample at 4 deg.C, centrifuging at 300rpm for 5min, sucking supernatant and white intermediate layer, and placing in sterilized 50mL centrifuge tube;
(4) washing the precipitate with 10mL precooled 0.1mol/L PBS buffer solution, centrifuging at 4 deg.C and 300rpm for 5min, collecting supernatant and white intermediate layer, repeating the process for 2 times, and mixing with the collected fermented grains sample;
(5) centrifuging all supernatants at 4 deg.C and 12000 rpm for 3 min, discarding the supernatant, and collecting cell precipitate;
(6) washing the cell precipitate with 10mL precooled 0.1mol/L PBS buffer solution, centrifuging at 4 deg.C and 12000 rpm for 3 min, and collecting the precipitate, and repeating the process for 2 times;
(7) filtering the collected precipitate, and freeze-drying at-80 deg.C or directly extracting DNA or RNA;
step two: extracting total RNA of a large-batch fermented grain sample:
(1) fully grinding the pre-treated fermented grains in liquid nitrogen until the cells of the fermented grains are completely broken, wherein the liquid nitrogen submerges the fermented grains;
(2) weighing 0.1g of ground large-batch fermented grains sample, placing the sample in a 1.5mL Eppendorf tube without RNase, adding 750 mu L of STE solution and 750 mu L of water-saturated phenol, and uniformly mixing by oscillation;
(3) heating the mixed large-batch fermented grains in 65 deg.C water bath for 10min, and fully shaking for 1 time every 2 min;
(4) placing the heated large-batch fermented grains sample in 12000 rpm, centrifuging at 4 ℃ for 5min, and then transferring the supernatant into a new 1.5mL RNase-free Eppendorf tube;
(5) 1/10 volumes of a mixture of 3mol/L sodium acetate with pH 5.2 and 200. mu.L acid phenol, chloroform and isoamyl alcohol were added to the supernatant, wherein the acid phenol: chloroform: isoamyl alcohol = 25: 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL Eppendorf tube without RNase, and extracting until no intermediate protein layer exists;
(6) and adding 200 mu L of chloroform-isoamyl alcohol mixed solution into the supernatant, wherein the ratio of chloroform: isoamyl alcohol = 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL RNase-free Eppendorf tube, and extracting until no layering exists;
(7) adding isopropanol with volume 1 time of that of the supernatant, standing at room temperature for 10min, centrifuging at 12000 rpm at 4 ℃ for 10min, and discarding the supernatant;
(8) washing the precipitate with 75% ethanol for 2 times, air drying at room temperature, adding ddH2O20 ~ 30 mu L, fully dissolving the precipitate;
(9) detecting the quality of RNA through electrophoresis (see figure 1), and simultaneously, measuring the concentration of RNA through Nanodrop;
step three: removing DNA in total RNA of a large-batch fermented grain sample:
(1) taking 10 mu g of total RNA, adding 3 ~ 5U of DNase1 into 100 mu L of reaction system, and reacting for 30 minutes at 37 ℃;
(2) the reaction system was supplemented with 600. mu.L of ddH2And adding 200 mu L of mixed solution of acid phenol, chloroform and isoamylol, wherein the acid phenol: chloroform: isoamyl alcohol = 25: 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL Eppendorf tube without RNase, and extracting until no intermediate protein layer exists;
(3) and (3) mixing 200 mu L of chloroform and isoamylol in the supernatant, wherein the ratio of chloroform: isoamyl alcohol = 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL Eppendorf tube without RNase, and extracting until no phenol layer exists;
(4) adding 1/10 volume of 3mol/L sodium acetate with pH of 5.2 and 2 times volume of anhydrous ethanol into the supernatant, standing at-70 deg.C for more than 1 hr, centrifuging at 12000 rpm at 4 deg.C for 10min, and removing supernatant;
(5) washing the precipitate with 75% ethanol for 2 times, air drying at room temperature, adding ddH2O 10 μ L, and dissolving the precipitate;
(6) agarose electrophoresis detection, and Nanodrop determination of RNA quality.
The 0.1mol/L PBS buffer solution comprises a stock solution and a diluent, wherein the stock solution: 34.0g of potassium dihydrogen phosphate is weighed and dissolved in 500mL of distilled water, the pH value is adjusted by 175 mL of 1mol/L sodium hydroxide solution, and the solution is diluted to 1000mL by the distilled water and then stored in a refrigerator; diluting liquid: the stock solution (1.25 mL) was diluted to 1000mL with distilled water, and the solution was dispensed into suitable containers and autoclaved at 121 ℃ for 15 min, and the final pH of the PBS buffer was 7.2.
The STE solution is: 0.3mol/L of sucrose; Tris-HCl 25mmol/L at pH 8.0; EDTA 25mmol/L at pH 8.0; and (5) sterilizing for later use.
Example 2
The method provided by the invention is used for analyzing the structure and succession rule of the microbial community of the Niuban mountain Erguotou fermentation. In the establishing process of the method, a pre-test is carried out in advance, according to the fermentation process of the Niubashan mountain double-pan head, the sampling positions are the central positions and the edge positions of the upper part, the middle part and the lower part of a ground cylinder, 300g of the second-batch fermented grains are taken at 0, 3, 5, 7, 9, 13, 19, 23 and 28 th time, the sampling position is the position where the center of the fermentation ground cylinder is 50cm away from the cylinder bottom, the second-batch fermented grains are placed in an aseptic sealed bag to be uniformly mixed, and the mixture is sent to a laboratory for later use within 10 min.
A method for rapidly and efficiently extracting RNA from fermented grains comprises the following steps:
the method comprises the following steps: pre-treating a sample of fermented grains in the second crop:
(1) weighing 1g of a second fermented grain sample, and suspending with 10mL of sterilized and precooled 0.1mol/L PBS buffer solution;
(2) adding four steel balls with the diameter of 1mm into the second fermented grain sample, and carrying out vortex oscillation treatment for 5min until the thalli are completely dispersed;
(3) placing the two-batch fermented grains sample after oscillation treatment at 4 ℃, centrifuging at 300rpm for 5min, sucking the supernatant and the white middle layer, and placing in a sterilized 50mL centrifuge tube;
(4) washing the precipitate with 10mL precooled 0.1mol/L PBS buffer solution, centrifuging at 4 deg.C and 300rpm for 5min, collecting supernatant and white intermediate layer, repeating the process for 2 times, and mixing with the collected second fermented grain sample;
(5) centrifuging all supernatants at 4 deg.C and 12000 rpm for 3 min, discarding the supernatant, and collecting cell precipitate;
(6) washing the cell precipitate with 10mL precooled 0.1mol/L PBS buffer solution, centrifuging at 4 deg.C and 12000 rpm for 3 min, and collecting the precipitate, and repeating the process for 2 times;
(7) filtering the collected precipitate, and freeze-drying at-80 deg.C or directly extracting DNA or RNA;
step two: extracting total RNA of a second fermented grain sample:
(1) fully grinding the pre-treated second fermented grain sample in liquid nitrogen until the cells of the second fermented grain sample are completely broken, based on submerging the large fermented grain in the liquid nitrogen;
(2) weighing 0.1g of ground second-batch fermented grains sample, placing the ground second-batch fermented grains sample in a 1.5mL Eppendorf tube without RNase, adding 750 mu L of STE solution and 750 mu L of water-saturated phenol, and uniformly mixing the two samples by oscillation;
(3) heating the mixed second batch of fermented grains in 65 deg.C water bath for 10min, and fully shaking for 1 time every 2 min;
(4) placing the heated second batch of fermented grains sample in 12000 rpm, centrifuging at 4 ℃ for 5min, and then transferring the supernatant into a new 1.5mL Eppendorf tube without RNase;
(5) 1/10 volumes of a mixture of 3mol/L sodium acetate with pH 5.2 and 200. mu.L acid phenol, chloroform and isoamyl alcohol were added to the supernatant, wherein the acid phenol: chloroform: isoamyl alcohol = 25: 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL Eppendorf tube without RNase, and extracting until no intermediate protein layer exists;
(6) and adding 200 mu L of chloroform-isoamyl alcohol mixed solution into the supernatant, wherein the ratio of chloroform: isoamyl alcohol = 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL RNase-free Eppendorf tube, and extracting until no layering exists;
(7) adding isopropanol with volume 1 time of that of the supernatant, standing at room temperature for 10min, centrifuging at 12000 rpm at 4 ℃ for 10min, and discarding the supernatant;
(8) washing the precipitate with 75% ethanol for 2 times, air drying at room temperature, adding ddH2O20 ~ 30 mu L, fully dissolving the precipitate;
(9) detecting the quality of RNA through electrophoresis (see figure 1), and simultaneously, measuring the concentration of RNA through Nanodrop;
step three: removing DNA in total RNA of the second fermented grain sample:
(1) taking 10 mu g of total RNA, adding 3 ~ 5U of DNase1 into 100 mu L of reaction system, and reacting for 30 minutes at 37 ℃;
(2) the reaction system was supplemented with 600. mu.L of ddH2And adding 200 mu L of mixed solution of acid phenol, chloroform and isoamylol, wherein the acid phenol: chloroform: isoamyl alcohol = 25: 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL Eppendorf tube without RNase, and extracting until no intermediate protein layer exists;
(3) and (3) mixing 200 mu L of chloroform and isoamylol in the supernatant, wherein the ratio of chloroform: isoamyl alcohol = 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL Eppendorf tube without RNase, and extracting until no phenol layer exists;
(4) adding 1/10 volume of 3mol/L sodium acetate with pH of 5.2 and 2 times volume of anhydrous ethanol into the supernatant, standing at-70 deg.C for more than 1 hr, centrifuging at 12000 rpm at 4 deg.C for 10min, and removing supernatant;
(5) washing the precipitate with 75% ethanol for 2 times, air drying at room temperature, adding ddH2O 10 μ L, and dissolving the precipitate;
(6) agarose electrophoresis detection, and Nanodrop determination of RNA quality.
The 0.1mol/L PBS buffer solution comprises a stock solution and a diluent, wherein the stock solution: 34.0g of potassium dihydrogen phosphate is weighed and dissolved in 500mL of distilled water, the pH value is adjusted by 175 mL of 1mol/L sodium hydroxide solution, and the solution is diluted to 1000mL by the distilled water and then stored in a refrigerator; diluting liquid: the stock solution (1.25 mL) was diluted to 1000mL with distilled water, and the solution was dispensed into suitable containers and autoclaved at 121 ℃ for 15 min, and the final pH of the PBS buffer was 7.2.
The STE solution is: 0.3mol/L of sucrose; Tris-HCl 25mmol/L at pH 8.0; EDTA 25mmol/L at pH 8.0; and (5) sterilizing for later use.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A method for rapidly and efficiently extracting RNA from fermented grains is characterized by comprising the following steps:
the method comprises the following steps: sample pretreatment:
(1) weighing 1g of solid sample, and suspending with 10mL of sterilized and precooled 0.1mol/L PBS buffer solution;
(2) adding four steel balls with the diameter of 1mm into the sample, and carrying out vortex oscillation treatment for 5min until the thalli are completely dispersed;
(3) placing the sample after the oscillation treatment at 4 ℃, centrifuging at 300rpm for 5min, sucking the supernatant and the white middle layer, and placing the supernatant and the white middle layer in a sterilized 50mL centrifuge tube;
(4) washing the precipitate with 10mL precooled 0.1mol/L PBS buffer solution, centrifuging at 4 deg.C and 300rpm for 5min, collecting supernatant and white intermediate layer, repeating the process for 2 times, and mixing with the collected sample;
(5) centrifuging all supernatants at 4 deg.C and 12000 rpm for 3 min, discarding the supernatant, and collecting cell precipitate;
(6) washing the cell precipitate with 10mL precooled 0.1mol/L PBS buffer solution, centrifuging at 4 deg.C and 12000 rpm for 3 min, and collecting the precipitate, and repeating the process for 2 times;
(7) filtering the collected precipitate, and freeze-drying at-80 deg.C or directly extracting DNA or RNA;
step two: extraction of total RNA of the sample:
(1) fully grinding the pretreated sample in liquid nitrogen until the sample cells are completely broken by taking the liquid nitrogen submerging the sample as a standard;
(2) weighing 0.1g of ground sample, placing the ground sample in a 1.5mL RNase-free Eppendorf tube, adding 750 mu L of STE solution and 750 mu L of water-saturated phenol, and uniformly mixing by oscillation;
(3) heating the mixed sample in 65 deg.C water bath for 10min, and fully shaking and mixing for 1 time every 2 min;
(4) the heated sample was centrifuged at 12000 rpm at 4 ℃ for 5min, and then the supernatant was transferred to a new 1.5mL RNase-free Eppendorf tube;
(5) 1/10 volumes of a mixture of 3mol/L sodium acetate with pH 5.2 and 200. mu.L acid phenol, chloroform and isoamyl alcohol were added to the supernatant, wherein the acid phenol: chloroform: isoamyl alcohol = 25: 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL Eppendorf tube without RNase, and extracting until no intermediate protein layer exists;
(6) and adding 200 mu L of chloroform-isoamyl alcohol mixed solution into the supernatant, wherein the ratio of chloroform: isoamyl alcohol = 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL RNase-free Eppendorf tube, and extracting until no layering exists;
(7) adding isopropanol with volume 1 time of that of the supernatant, standing at room temperature for 10min, centrifuging at 12000 rpm at 4 ℃ for 10min, and discarding the supernatant;
(8) washing the precipitate with 75% ethanol for 2 times, air drying at room temperature, adding ddH2O20 ~ 30 mu L, fully dissolving the precipitate;
(9) detecting the quality of RNA through electrophoresis, and simultaneously determining the concentration of RNA through Nanodrop;
step three: removal of DNA from total RNA in the sample:
(1) taking 10 mu g of total RNA, adding 3 ~ 5U of DNase1 into 100 mu L of reaction system, and reacting for 30 minutes at 37 ℃;
(2) the reaction system was supplemented with 600. mu.L of ddH2And adding 200 mu L of mixed solution of acid phenol, chloroform and isoamylol, wherein the acid phenol: chloroform: isoamyl alcohol = 25: 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL Eppendorf tube without RNase, and extracting until no intermediate protein layer exists;
(3) and (3) mixing 200 mu L of chloroform and isoamylol in the supernatant, wherein the ratio of chloroform: isoamyl alcohol = 24: 1, oscillating uniformly, centrifuging at 12000 rpm at 4 ℃ for 5min, transferring the supernatant into a new 1.5mL Eppendorf tube without RNase, and extracting until no phenol layer exists;
(4) adding 1/10 volume of 3mol/L sodium acetate with pH of 5.2 and 2 times volume of anhydrous ethanol into the supernatant, standing at-70 deg.C for more than 1 hr, centrifuging at 12000 rpm at 4 deg.C for 10min, and removing supernatant;
(5) washing the precipitate with 75% ethanol for 2 times, air drying at room temperature, adding ddH2O 10 μ L, and dissolving the precipitate;
(6) agarose electrophoresis detection, and Nanodrop determination of RNA quality.
2. The method for rapidly and efficiently extracting RNA from fermented grains according to claim 1, wherein the sample is fermented grains from large batches or fermented grains from second batches in the fermentation process of white spirit.
3. The method for rapidly and efficiently extracting RNA from fermented grains according to claim 1, wherein the 0.1mol/L PBS buffer solution comprises a stock solution and a diluent, wherein the stock solution: 34.0g of potassium dihydrogen phosphate is weighed and dissolved in 500mL of distilled water, the pH value is adjusted by 175 mL of 1mol/L sodium hydroxide solution, and the solution is diluted to 1000mL by distilled water and then stored in a refrigerator; diluting liquid: the stock solution (1.25 mL) was diluted to 1000mL with distilled water, and the solution was dispensed into suitable containers and autoclaved at 121 ℃ for 15 min, and the final pH of the PBS buffer was 7.2.
4. The method for rapidly and efficiently extracting RNA from fermented grains according to claim 1, wherein the STE solution comprises: 0.3mol/L of sucrose; Tris-HCl 25mmol/L at pH 8.0; EDTA 25mmol/L at pH 8.0; and (5) sterilizing for later use.
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