Method for extracting total RNA from fermented grains used for Chinese liquor fermentation

A technology for fermenting wine and liquor, applied in the biological field, can solve the problems of only targeting a certain type or a certain type of microorganisms, not being suitable for extracting community microorganisms, and poor wall breaking effect, achieving good extraction rate, wide application range, The effect of the simple extraction process

Active Publication Date: 2016-10-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the SDS method has a better effect on the total RNA extraction of prokaryotic microorganisms, but for eukaryotic microorganisms with cell walls, the effect of SDS on breaking the wall is very poor, so it cannot be used in the total RNA extraction of microorganisms with both eukaryotic and prokaryotic communities
However, the CTAB method needs to be preheated. If the temperature is too high, the RNA degrading enzyme will become active and the RNA will be degraded. At the same time, the CTAB method is easy to use, and the operation is troublesome. LiCl is usually used together.
The hot boric acid method not only needs to add protease, KCl and other matching reagents, but also requires you to preheat. If the temperature is too high, the RNA degrading enzyme will become active and the RNA will be degraded.
[0005] In addition, these methods are all aimed at a single type or a certain kind of microorganisms, and are not suitable for extracting community microorganisms, especially when eukaryotic and prokaryotic microorganisms are mixed together

Method used

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  • Method for extracting total RNA from fermented grains used for Chinese liquor fermentation
  • Method for extracting total RNA from fermented grains used for Chinese liquor fermentation

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Experimental program
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Effect test

Embodiment 1

[0066] The microbial total RNA was extracted from the fermented grains of Chinese Maotai-flavor liquor according to the following method:

[0067] (1) Take 25g sample and dissolve it in 50mL sterile 0.1mol / L PBS (42.3ml 1mol / LNaH 2 PO 4 and 57.7ml1mol / LNa 2 HPO 4 , dilute to 1L with deionized water, then sterilize at 121°C for 15 minutes) to suspend in the buffer, add 5g of glass beads, and vortex for 2-3 minutes;

[0068] (2) Centrifuge at 500rpm for 5min, get the supernatant, wash the precipitation three times with PBS buffer, collect all the supernatant after centrifugation; remove the remaining impurities in the fermented wine grains except microorganisms by step (1) and step (2) , including solid grains and liquid-soluble polysaccharides and heteroacids;

[0069] (3) Centrifuge the supernatant at 12000rpm for 15min, discard the supernatant, cover the tube cap, and freeze in liquid nitrogen;

[0070] (4) Prepare the extraction mixture (composed of 20 parts of sodium l...

Embodiment 2

[0081] The microbial total RNA of fermented grains of Chinese Fen-flavor liquor was extracted according to the following method:

[0082] (1) Dissolve 25g of sample in 50mL of sterile 0.1mol / L PBS buffer and suspend, add 5g of glass beads, and vortex for 2-3min;

[0083] (2) Centrifuge at 550rpm for 4min, get the supernatant, wash the precipitation three times with PBS buffer solution, collect all the supernatant after centrifugation; remove the remaining impurities in the fermented wine grains except microorganisms by step (1) and step (2) , including solid grains and liquid-soluble polysaccharides and heteroacids;

[0084] (3) Centrifuge the supernatant at 11,000 rpm for 8 minutes, discard the supernatant, cover the tube cap, and freeze in liquid nitrogen;

[0085] (4) Prepare the extraction mixture (composed of 15 parts of sodium laurate extraction buffer, 25 parts of Trizol, 0.5 parts of mercaptoethanol and 1.5 parts of dithiothreitol), mix gently, and place on ice;

[0...

Embodiment 3

[0096] The microbial total RNA of Chinese Luzhou-flavor liquor fermented grains was extracted according to the following method:

[0097](1) Dissolve 25g of sample in 50mL of sterile 0.1mol / L PBS buffer and suspend, add 5g of glass beads, and vortex for 2-3min;

[0098] (2) Centrifuge at 450rpm for 8min, get the supernatant, wash the precipitation three times with PBS buffer, collect all the supernatant after centrifugation; remove the remaining impurities in the fermented wine grains except microorganisms by step (1) and step (2) , including solid grains and liquid-soluble polysaccharides and heteroacids;

[0099] (3) Centrifuge the supernatant at 13,000 rpm for 12 minutes, discard the supernatant, cover the tube cap, and freeze in liquid nitrogen;

[0100] (4) Prepare the extraction mixture (composed of 25 parts of sodium laurate extraction buffer, 15 parts of Trizol, 1.5 parts of mercaptoethanol and 0.5 parts of dithiothreitol), mix gently, and place on ice;

[0101] (5) ...

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Abstract

The invention discloses a method for extracting total RNA from fermented grains used for Chinese liquor fermentation, and belongs to the biotechnical field. The total DNA is rapidly extracted from microbial communities in 2h against the particularity of a Chinese liquor fermented grain sample through combining a sodium laurate extraction technology and a guanidinium isothiocyanate-phenol-chloroform extraction technology. The microbial total RNA extracted through using the method has the advantages of high output, good integrity, good purity, and effective extraction of eukaryotic and prokaryotic microbes from the sample.

Description

technical field [0001] The invention relates to a method for extracting total RNA from Chinese liquor fermented grains, belonging to the field of biotechnology. Background technique [0002] Under the current technical conditions, more than 95% of microorganisms cannot be cultivated, which is the biggest obstacle in the research of traditional microbial ecology in revealing the structure of microbial communities in nature, ecological functions and their relationships. In recent years, great success has been achieved in studying environmental microorganisms at the mRNA level through the method of metatranscriptomics, and the results obtained are more accurate and reliable. With the in-depth research on the structure and function of community microorganisms, it is necessary to extract the total RNA of all microorganisms in a certain ecosystem as the basic requirement for analyzing the structure and function of community microorganisms. [0003] At present, the closest method ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 杜海徐岩宋哲玮
Owner JIANGNAN UNIV
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