Tropical plant polysaccharide and polyphenol small RNA extraction method

Abstract

The present invention discloses a tropical plant polysaccharide and polyphenol small RNA extraction method, which comprises: sampling tropical plant leaves, freezing in liquid nitrogen, and storing at a temperature of -80 DEG C; preparing small RNA extraction reagents, wherein the reagents comprise a CTAB lysis buffer solution, a PEG8000 precipitation buffer solution, an EDC cross-linking buffer solution, a miRNA Northern blotting hybridization buffer solution, 7% dPAGE, and Washing Buffer; according to small RNA extraction pretreatment, Trizol extraction, fractional precipitation of high molecular weight RNA, and small RNA precipitation, extracting small RNA; according to 17% dPAGE separation, trarsmembrane and cross-linking, miRNA hybridization and washing of membrane, small RNA hybridization and washing of membrane, and exposure and storage, achieving RNA hybridization; carrying out miRNA stem-loop RT-PCR amplification; and carrying out extraction and detection analysis on polysaccharide and polyphenol plant small RNA. According to the present invention, the defects in the current plant small RNA extraction method are overcome, characteristics of fastness, stability and easy operation are provided, rapid extraction and detection analysis can be performed on miRNA and siRNA, and broad application prospects are provided.

Description

technical field [0001] The invention belongs to the technical field of RNA extraction, and in particular relates to a method for extracting tropical plant polysaccharide polyphenol small RNA. Background technique [0002] Plant small RNAs are negative regulators that regulate gene expression, DNA and histone methylation and resistance to exogenous genetic factors such as viruses, transposons or transgenes at the transcriptional and post-transcriptional levels according to sequence specificity. According to the biosynthesis characteristics of small RNA, plant small RNA is mainly divided into two categories: plant small interfering RNA (siRNA, small / short interfering RNA) and microRNA (miRNA, miRNA). The length of the two is about 20-25nt, both are processed by the RNAe III-like endonuclease Dicer (or Dicer-like protein), and their formation is both related and different. MicroRNA (or miRNA) is a single-stranded small molecule RNA with a length of 20-24nt, which is produced b...

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Problems solved by technology

[0010] The purpose of the embodiments of the present invention is to provide a method for extracting small RNA polysaccharides and polyphenols from tropical plants, which aims to solve the problem of the change of mobility caused by the combination of polysaccharides and polyphenols impurities in plants and small RNAs

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