A method for extracting microbial total rna in forest soil and litter

Abstract

The invention discloses a method for extracting microbial total RNA in forest soil and litter, which comprises the following steps: 1, carrying out freeze-drying on a forest soil or litter sample, grinding, and uniformly mixing; 2, adding pyrocarbonic acid diethyl ester treating water into the powdered sample, and standing over night at -70 DEG C; 3, adding glass beads, hexadecyl trimethyl ammonium bromide buffer, lauryl sodium sulfate, diatomite and phenol / chloroform / isoamyl alcohol, and uniformly mixing; 4, severely shaking the mixed liquid, and centrifuging to obtain the supernate; 5, adding guanidinium isothiocyanate into the supernate, and centrifuging to obtain the supernate; 6, adding chloroform / isoamyl alcohol into the supernate, and centrifuging to obtain the supernate; 7, addingpolyethylene glycol 6000 into the supernate, standing, centrifuging, and removing the supernate; 8, washing, dissolving, and carrying out DNA enzyme digestion to obtain an RNA solution; and 9, detecting the integrality of the RNA through agarose gel electrophoresis, and using a nucleic acid protein determinator to detect the concentration and purity of the RNA. The RNA extracted by using the method has high yield, good integrality and better purity.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for extracting total RNA of microorganisms, in particular to a method for extracting total RNA of microorganisms in forest soil and litter. This method is mainly suitable for the extraction of total microbial RNA in samples during the research of microbial functional gene transcriptomics in forest soil and litter in scientific research, teaching, environmental protection and other departments. 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. With the continuous penetration of molecular biology theory and technology in microbial ecology research, the method of directly analyzing the total microbial DNA isolated ...

AI Technical Summary

Problems solved by technology

However, forest litter and soil are rich in humic acid, polysaccharides, polyphenols and other substances, so that the purity of the extracted microbial total RNA is not high, and RNase is widely present in the environment, and RNA is easily degraded by it, which brings great inconvenience to RNA extraction. a lot of difficulties

There are a few commercial companies that have developed soil RNA extraction kits, but they are expensive and have strong selectivity for soil samples, especially when extracting total microbial RNA from forest litter, the RNA yield is low and the effect is unstable

However, most of the soil microbial total RNA yields obtained by the hand-held method are not high, and the humic acid pollution is serious, which will interfere with the smooth progress of downstream experiments.

In addition, the current methods for extracting total RNA from environmental microorganisms are mainly for environmental samples such as soil, compost, water, and atmosphere, and there is still a lack of effective extraction methods for total RNA of microorganisms in forest litter.

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