DsRNA in organism or tissue and method for extracting dsRNA

Abstract

The invention relates to dsRNA in an organism or tissue and a method for extracting the dsRNA. The method comprises the following steps: pre-treating, centrifuging to obtain supernate, carrying out chromatography on the supernate, carrying out segmented elution, and collecting eluent for post-treatment. According to the method, organic reagent treatment and cellulose column chromatography are combined, firstly, an organism or tissue is treated to be broken, and nucleic acid substances are released into a liquid phase; and by means of reversible adsorption of filler in cellulose column chromatography on liquid-phase medium-concentration nucleic acid substances, dsRNA and other nucleic acid substances can be effectively separated through segmented elution, especially interference of DNA degradation substances can be remarkably reduced, and the purity of the final product dsRNA can be improved.

Description

technical field [0001] The invention relates to the technical field of dsRNA extraction, in particular to a dsRNA in organisms or tissues and a method for extracting the dsRNA. Background technique [0002] It is difficult to detect the existence of a large amount of double-stranded RNA (double-stranded RNA, dsRNA) in normal animals and plants. Any situation that leads to the formation of dsRNA in normal plants will cause the silence of the corresponding gene, which is the dsRNA cutting enzyme of the host ( Dicer enzyme) cutting results, so the effective expression of various genes in normal animals and plants requires a strict mechanism to prevent the formation of dsRNA, which is RNA interference (RNA interference, RNAi) in organisms. Utilizing the principle and technology of RNAi, with the help of transgenes or nano-carriers or other means, it can specifically reduce or shut down the expression of host or pathogen genes. This technology has achieved certain results in the...

AI Technical Summary

Problems solved by technology

[0004] The current dsRNA extraction methods are: (1) TRIzol method, that is, the total RNA is first extracted and then digested with DNase and RNase to obtain dsRNA, but the quality of dsRNA extracted by this method is poor and the efficiency is very low

(2) LiCl precipitation method, that is, extract the total RNA first and then use LiCl to perform density gradient centrifugation to obtain dsRNA, but the quality of the dsRNA extracted by this method is also relatively poor, and more importantly, the operation is cumbersome and the price of the reagent too expensive

[0005] However, although these conventional methods can extract dsRNA, the operation process is still relatively cumbersome, and more time is consumed in the extraction process, and more dsRNA will be lost during multiple centrifugation suspensions in centrifuge tubes , the system is unstable, it is easy to cause the failure of the dsRNA extraction experiment, which is not conducive to the normal development of the experiment, and slows down the progress of the experiment; and during the extraction process, a large amount of interfering substances will be generated due to the degradation of DNA, resulting in a low concentration of dsRNA in the extract. And affect the final product purity and extraction recovery

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