葡萄总RNA提取方法的研究

针对葡萄组织中多酚、多糖类物质含量较高的特点,比较了改进SDS/酚法、异硫氰酸胍法和市售总RNA提取试剂盒等不同的提取方法,3种改进方法均能从葡萄幼嫩叶片中提取到总RNA。其中改进的SDS/酚法能有效抑制酚类物质和多糖对总RNA提取的影响,能够从成熟的叶片和完熟果实的果皮中获得质量高、完整性好的总RNA,28SrRNA亮度约为18SrRNA的2倍,A260/A280介于1.8～2.0之间,A260/A230大于2.0,且总RNA产率高,其中幼叶总RNA产率为261.20μg/g,成熟叶产率为191.40μg/g,完熟果皮产率为31.50μg/g,完全适于进行DDRT-PCR、Northernbolt和cDNA文库构建等研究。且该方法具有快速、简单、有效的特点。

An Improved Method for Rapidly Extracting Total RNA from Vitis

Extracting high-quality RNA from grapevine tissues are particularly difficult due to high levels of polyphenolics and polysaccharides,or other compounds that bind and/or coprecipitate with RNA.The modified SDS/phenol,guanidinum thiocyanate and commercial total RNA extraction kit were applied to isolate high purity and integrity RNA from grapevine.The normal component of the total RNA isolation method is the addition of polyvinylpyrrolidone(PVP)andβ-mercaptoetha-hol in the extraction buffer to SDS/phenol and guanidium thiocyanate,and exchanging Trizol in Kit with guanidium thio-cyanate.These methods all can obtain total RNA from young leaf of grapevine,but only the modified SDS/phenol method ex-tract integrity and undegraded RNA from mature leaf and ripe fruit skin.Following this efficient procedure,we routinely ob-tained191.40～261.20μg /g total RNA from leaf and31.50μg /g from ripe fruit skin.The ratio of A 260 /A 280 =1.8～2.0.The RNA is suitable for DDRT-PCR,cDNA library construction and Northern hybridization.