Extraction method of RNA (ribonucleic acid) of rape seeds

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an extraction method of an RNA (ribonucleic acid) of rape seeds. The method comprises the following steps of: preheating a hot borate buffer solution to 65 DEG C; quickly freezing the rape seeds with liquid nitrogen, grinding into powder, adding the preheated buffer solution, and continuously grinding to form homogenate; incubating, precipitating proteins with potassium chloride, and performing centrifugation, impurity removal and other steps to obtain general total RNA. The total RNA of the rape seeds extracted by the method has complete quality and no remarkable degrading phenomenon, the OD260 / OD280 and OD260 / OD230 are at about 2.0, other impurity pollution, such as proteins and salt ions, does not exist in the sample, and the product production rate is more than 40mu g / 100mg, which is in accordance with the requirement of cDNA library construction.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to an RNA extraction method suitable for rapeseeds, which uses hot borate method to extract high-quality and high-purity rapeseed total RNA, so as to meet the requirements of next-generation sequencing library construction and seed development Gene cloning, expression analysis and verification of RNA quality requirements. Background technique [0002] The content of protein, oil and fiber in rapeseed is high, especially in mature seeds, while the content of RNA is relatively low. In addition, the seed coat is rich in metabolites such as pigments, tannins, and polyphenols. The existence of these substances seriously affects the yield of RNA extraction, and will directly interfere with subsequent experiments such as library construction and reverse transcription. Therefore, how to extract RNA with good integrity and high purity is the premise of studying rape s...

AI Technical Summary

Problems solved by technology

At present, a large number of commercialized kits have been applied to the extraction of plant total RNA, such as the kits of well-known molecular reagent companies such as Invitrogen and Fermentas, whose RNA extraction quality is intact, but the price is relatively expensive; Takara’s RNA extraction kit is moderately priced, However, the extracted RNA contains high protein and salt ions. The presence of these impurities will affect the efficiency of library construction, which may lead to the failure of library construction. It can only be used for reverse transcription and other experiments, and cannot meet the requirements of library construction.

Many researchers have discussed rapeseed RNA extraction methods, and proposed CTAB method, SDS method, etc. The extracted RNA can be successfully used for reverse transcription, etc., but whether it can be applied to cDNA library construction has not been reported yet.

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