Method for efficiently extracting microorganism total RNA (Ribonucleic Acid) from anaerobic granular sludge

An anaerobic granular sludge and microorganism technology, which is applied in the field of environmental molecular biology, can solve the problems that activated sludge is not suitable, is not suitable for activated sludge, cannot reflect the real state, etc., to promote stability and impurity residue. The effect of fewer and simplified extraction steps

Active Publication Date: 2014-04-02
吉林梅花氨基酸有限责任公司
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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

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  • Method for efficiently extracting microorganism total RNA (Ribonucleic Acid) from anaerobic granular sludge
  • Method for efficiently extracting microorganism total RNA (Ribonucleic Acid) from anaerobic granular sludge
  • Method for efficiently extracting microorganism total RNA (Ribonucleic Acid) from anaerobic granular sludge

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Embodiment 1

[0049] Activated granular sludge samples were taken from the sewage treatment plant of Futian Pharmaceutical Co., Ltd., Yucheng City, Shandong Province.

[0050] Take 10mL of activated sludge and centrifuge at 8000r / min at 4°C for 5min in a 15mL sterilized centrifuge tube. After discarding the supernatant, add 3g of sterilized glass beads (diameter 2-3mm) and 1.4mL of stock solution I into the tube. Tighten the cap of the centrifuge tube and place it in a vortex mixer for 5 minutes to deflocculate and wash the sludge to remove humic acid. Centrifuge at 8000r / min at 4°C for 5 minutes. Discard the supernatant. Repeat the treatment once and buffer with phosphate. The sludge was washed once with liquid PBS, and the pellet was used for cell lysis. Take a certain amount of pretreated sludge sample and add 100 μL common storage solution II, incubate at 37°C for 10 minutes, add 1 mL TRIzol, vortex for 5 minutes, centrifuge at 10,000 r / min for 10 minutes, take the supernatant and add 2...

Embodiment 2

[0055] The activated granular sludge samples were taken from the sewage treatment plant of Baolingbao Co., Ltd., Yucheng City, Shandong Province.

[0056] Take 10mL of activated sludge and centrifuge at 8000r / min at 4°C for 5min in a 15mL sterilized centrifuge tube. After discarding the supernatant, add 3g of sterilized glass beads (diameter 2-3mm) and 1.4mL of stock solution I into the tube. Tighten the cap of the centrifuge tube and place it in a vortex mixer for 5 minutes to deflocculate and wash the sludge to remove humic acid. Centrifuge at 8000r / min at 4°C for 5 minutes. Discard the supernatant. Repeat the treatment once and buffer with phosphate. The sludge was washed once with liquid PBS, and the pellet was used for cell lysis. Take a certain amount of pretreated sludge sample and add 100 μL common storage solution II, incubate at 37°C for 10 minutes, add 1 mL TRIzol, vortex for 5 minutes, centrifuge at 10,000 r / min for 10 minutes, take the supernatant and add 200 μL c...

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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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Application Information

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IPC IPC(8): C12N15/10
Inventor 姬丹丹臧立华薛嵘李肖玲
Owner 吉林梅花氨基酸有限责任公司
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