芸芥柱头总RNA提取方法比较研究

Comparison and Improvement of Different Extracting Total RNA Methods from Stigmas in E.Sativa Mill.

为了确立一种适合于芸芥柱头总RNA的提取方法,以芸芥柱头为材料,在常规异硫氰酸胍法的基础上提取和纯化总RNA。结果表明,采用常规异硫氰酸胍法提取的芸芥柱头总RNA,28SRNA和18SRNA条带的亮度之比接近于1∶1,而不是所要求的2∶1,产生了明显的降解。改进异硫氰酸胍法提取的芸芥柱头总RNA,28SRNA和18SRNA条带的亮度之比接近于2∶1,提取的RNA完整性好,但存在痕量DNA污染。用DNase降解痕量DNA,发现DNA去除较干净,且RNA没有发生降解,可作为反转录底物进行第一链cDNA合成。确立了一种适合于芸芥柱头总RNA的提取与纯化方法,为芸芥自交亲和基因的克隆及功能分析奠定了基础。

This study was conducted to develop a suitable method for extracting total RNA trom stigma in E. Sativa Mill. By using the improved guanidine thioeyanate method, total RNA from Yunjie stigmas were extracted and pured. The results showed that the total RNA were in good integrity and high purity, which demonstrated that RNA samples were free of contamination of protein, DNA, polysaccharide and polyphenol, and were applicable to cDNA synthesis, DDRT-PCR, northern blotting and so on. This method was simple, economic, and of stable performance, good repeatability as well as suitable for extracting total RNA from many materials.