Extraction method for RNA (ribonucleic acid) of secondary dormancy seeds of mature rapes

Abstract

The invention belongs to the field of plant biotechnology and particularly relates to a rapid extraction method of high-quality RNA (ribonucleic acid) of secondary dormancy seeds of mature rapes. The method comprises the following steps: selecting a total RNA extracting reagent of RNA plant plus reagent plant, inhibiting RNA enzymatic activity and preventing phenolic compounds from being oxidized by using beta-mercaptoethanol, removing the influence of polysaccharides to RNA by applying sodium acetate, and reducing the extracting times. According to the method, the extraction of RNA of the secondary dormancy seeds of the rapes only needs 30min, the RNA quality is good and the obtained RNA is high in purity, and the method has the advantages that the using amount of the reagent is few, the economy and the saving are achieved, and the like, and can be successfully applied to subsequent mRNA separation, purification, database building and high-throughput transcriptome sequencing. According to the method, the important technical guaranteeing is provided for verifying molecular mechanism of a second dormancy seeds of rape, and simultaneously, the technical reference is provided for the extraction for RNA of mature dormancy seeds of other polysaccharide plants and polyphenols plants.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and in particular relates to the rapid extraction of high-quality RNA from secondary dormant seeds of mature rapeseed. Background technique [0002] RNA extraction technology is not only a basic experimental technique in molecular biology, but also a necessary prerequisite for molecular biology research. Only high-quality total RNA with high purity and good integrity can be used for RT-PCR, cDNA synthesis, RNA sequence analysis, mRNA purification for protein translation in vitro or construction of cDNA library, and mRNA differential display, high-throughput Subsequent molecular biology research such as transcriptome sequencing. [0003] Rapeseed is a fatty seed, which contains a lot of lipids, proteins, polysaccharides, vitamins and polyphenols, which seriously affects the extraction of RNA. Therefore, the extraction of total RNA from rapeseed is difficult, and the reported methods for extrac...

AI Technical Summary

Problems solved by technology

Because the old and healthy tissue of mature rapeseed is rich in RNase, and the physiological activity of the seed is greatly reduced after the seed enters a dormant state, it is difficult to guarantee the isolation effect and integrity of rapeseed RNA

However, using the existing methods for RNA extraction from fresh rapeseed or incompletely mature rapeseeds, there are disadvantages such as large reagent consumption and long extraction time (1-8.5 hours), especially if the extraction time is too long, it will directly increase RNA contamination. And the possibility of degradation, and eventually lead to extraction failure

Images