白菜总RNA的高效提取方法及常见问题分析

Analysis on Efficient Extraction Method and Common Problem of Totul from Chinese Cabbage

[目的]建立从白菜叶片组织中快速提取高质量RNA的方法，为进行LD—PCR、AnP、SRAP及其他分子生物学试验奠定基础。[方法]通过增加高速离心时间，用3％H2O2处理试验器皿等措施对Trizol试剂法进行优化，建立了一种简单、快捷、经济、高效的RNA提取方法。[结果]在1．5h左右可得到高质量的总RNA，经琼脂糖凝胶电泳、蛋白核酸分析仪检测，提取的总RNA具有清晰的28S、18S、5S3条带，且28S的宽度是18S的2倍左右，0D260／DD280比值为1．90—2．16。进一步以提取的总RNA为模板成功进行了单链cDNA的合成和LD—PCR。[结论]改良的Trizol试剂法所提取的RNA纯度和完整度极高，可用于LD—PCR、SRAP、基因克隆及表达分析等后续分子生物学试验。

[ Objective ] To develop a method for rapid extraction of total RNA from Chinese cabbage in order to lay the foundations for LDPCR,AFLP,SRAP and most molecular biological experiments. [ Method] The method of Trizol isolation of total RNA was modified to efficiently isolate high quality total RNA from Chinese cabbage leaves. By increasing high-speed centrifuge time, and immersing experimental containers with 3% H202. [ Result] High-quality total RNA was obtained about 1.5 h. The quality of total RNA was analyzed with agarose gel electrophoresis and nucleic acid and protein analyzer. The RNA obtained showed three bright bands in agarose gel electrophoresis 28 S, 18 S and 5 S. The band of 28 S was twice wider than that of 18 S, and the value of OD260/OD280 was 1.90 to 2.16. The RNA isolated by this method would be more competent for the first strand eDNA synthesis and LD-PCR. [ Conclusion ] Total RNA extracted by modified Trizol method had high purity quality and completeness, and can be used in LD-PCR,SRAP and most molecular biological experiments including gene cloning and expression analysis.