Method for extracting high-quality total RNA from mesenteric adipose tissues of grass carps

Abstract

The present invention discloses a method for extracting high-quality total RNA from mesenteric adipose tissues of grass carps. The method comprises the following steps: quick-freezing samples with liquid nitrogen, conducting lysis with a TRIzol reagent and mechanical homogenization, conducting centrifugation to remove a grease layer, chloroform-separating of a RNA-containing water phase, conducting isopropanol precipitation and 75% ethanol washing, etc. In a sample pre-processing stage, the sampling amount is increased to 30 mg to meet a requirement of RNA amount in a conventional molecular biology experiment; the samples are quick-frozen with the liquid nitrogen without manual grinding, the frozen samples are subjected to mechanical homogenization for a long time in the TRIzol reagent with a duration of 3 min, the occurrence of RNA degradation can be significantly reduced, at the same time, 2 centrifugal removals of the grease layer and other operation steps are increased, and purityand integrity of the obtained total RNA are higher. The extraction method effectively solves problems that grass carp adipose tissue RNA extraction is easy to degrade RNA and low in yield, and beside,the extraction method is also simple in operation, stable in a batch processing effect, can meet high-quality RNA requirement experiments such as transcriptome sequencing, and is also suitable for total RNA extraction of other fish adipose tissues.

Description

Technical field [0001] The invention relates to the field of fish RNA extraction, in particular to a method for extracting high-quality total RNA from grass carp mesenteric fat tissue. Background technique [0002] Animal adipose tissue is not only the body's energy storage, but also an active endocrine organ. The various hormones and cytokines secreted by it play an important role in the body's metabolic balance and immune response (Scherer, 2006; Wellen, Hotamisligil, 2003). In tissue function research, molecular level research is indispensable, among which RNA extraction and purification is a molecular biology experimental technique often involved. However, because adipose tissue is rich in oil and low in RNA abundance, it is often difficult to extract high-quality total RNA, which affects the efficient development of downstream experiments. [0003] At present, RNA extraction widely uses the classic acidic phenol-guanidine isothiocyanate-chloroform one-step extraction method (...

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Problems solved by technology

The test also found that the currently published optimization and improvement methods or strategies failed to stably obtain high-quality grass carp adipose tissue RNA

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