A method for efficiently extracting microbial total RNA from anaerobic granular sludge

Abstract

The invention belongs to the technical field of environment molecular biology and relates to a method for efficiently extracting microorganism total RNA (Ribonucleic Acid) from anaerobic granular sludge. The method comprises the following steps: fetching activated sludge, centrifuging, discarding supernatant, then adding sterilized glass beads and a stock solution I into a centrifugal tube, putting the centrifugal tube in a vortex mixer for beating after a tube cover of the centrifugal tube is screwed tightly, centrifuging to discharge supernatant, and washing by a phosphate buffer solution; adding a stock solution II and incubating; adding a TRIzol, centrifuging to obtain supernatant after vortex oscillation, adding chloroform, carrying out vortex oscillation, putting in a room temperature, centrifuging to obtain supernatant, adding isopropanol, putting in a temperature of -20 DEG C, centrifuging to discharge supernatanth, adding 75% ethanol to wash precipitates, centrifuging to discharge supernatant, air-drying the precipitates, dissolving nucleic acid precipitates by a stock solution III, adding DNase I (RNase free) and RNase inhibitors and adopting an RNA purifying reagent kit to purify the RNA. The steps for extracting the microorganism total RNA are simplified, and the microorganism total RNA is quickly and efficiently extracted from the anaerobic granular sludge.

Description

technical field [0001] The invention belongs to the technical field of environmental molecular biology, and in particular relates to a method for efficiently extracting total RNA from anaerobic granular sludge microorganisms. Background technique [0002] Activated granular sludge is a tea-brown floc formed by mixing bacteria-based microorganisms with suspended matter, colloidal substances, humus, etc. Among them, microorganisms are the main body of the wastewater biological treatment process, and their physiological status directly affects processing effect. Activated granular sludge has a high degree of biodiversity and extremely complex components. It not only contains heavy metals, humic acid and other organic pollutants, but also contains a large amount of ribonuclease (RNase). Therefore, it is difficult to extract RNA from activated sludge. In recent years, with the rapid development of molecular biology technology, the research on the relationship between the diversi...

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Problems solved by technology

[0003] Existing studies have shown that the research on microorganisms in activated sludge by means of molecular biology is mainly based on the DNA level, but the ecological analysis of microorganisms such as microbial diversity and abundance detected at the DNA level is not affected by viability, metabolic status, etc. The impact of environmental conditions and other factors cannot reflect the real state, while the analysis of microbial populations and gene expression at the RNA level can more truly reflect the status of the analysis, so the extraction of high-quality RNA is at the level of gene expression Necessary conditions for studying the changes of microbial flora and the expression of key regulatory genes in activated sludge during wastewater biological treatment

Regarding the extraction methods of RNA, a large number of research reports have been done at home and abroad, mainly including Trizol method, guanidine isothiocyanate method, phenol-SDS method, etc., but these methods can only be obtained from general bacteria, animals, plants and other materials. Extract better quality RNA, not suitable for activated sludge with complex components

The reported RNA extraction methods for environmental samples are mainly for soil, sediment, etc., but the characteristics of activated granular sludge are different from these environmental samples, not only has a large biomass, but also has bacteria and micelles, so these methods are not suitable for components complex activated sludge

Although foreign literature reports that freeze-thaw methods, bead milling methods, and kit methods can extract RNA from samples such as soil, sedimentary sludge, and activated sludge, these methods have their own shortcomings, and the freeze-thaw method is time-consuming. The bead mill method requires a special instrument bead mill, and the kit is expensive, and it is difficult to be used for the analysis of a large number of samples. In addition, there are few reports on the extraction method of RNA in activated sludge in China.

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