草鱼成熟脂肪细胞总RNA提取方法研究

摘要

RNA extraction is the basis of the molecular biology study, especially in reverse transcription PCR, Northern blot and RNA - Seq. Due to the high content of lipid in adipocytes of Ctenopharyngodon idellus, there are some difficulties to obtain high quality RNA. In this study, adipocytes were isolated from C. idellus mesenteric adipose tissues, and total RNA was extracted from these cells using kit with increasing the steps of centrifugation and extraction. The integrity, purity and quality were measured using agarose gel electrophoresis, nucleic acid quantitative analysis and gene amplification. The results showed that the A260 nn,/A280 nm ratio of RNA was 1.95 ~ 2. 00, the band of 28S and 18S was integrated, and the amplified bands of LPL and (3 - actin were clear. It was proved that high quality RNA of C. idellus adipocytes could be obtained with these modifications and suitable for common molecular biological studies.%获得高质量的RNA是逆转录聚合酶链式反应(RT-PCR)、Northern blot以及转录组测序(RNA-Seq)等分子生物学研究的基础,由于草鱼脂肪细胞中油脂含量较高,从中提取高质量RNA存在困难.本研究以草鱼(Ctenopharyngodon idellus)成熟脂肪细胞为材料,通过增加离心和抽提次数等方式对传统总RNA提取方法进行优化,采用琼脂糖凝胶电泳、核酸定量分析及基因扩增等方法验证所得RNA的完整性、纯度及质量.结果表明,经此方法提取的草鱼成熟脂肪细胞总RNA的A260nm/A280nm比值在1.95到2.00之间,28S和18S条带完整,草鱼LPL和β-actin基因扩增条带清晰.认为采用本方法获得的草鱼脂肪细胞RNA样品质量较高,能够用于后续分子生物学研究.