一种适用于RT-PCR的微藻总RNA提取方法

An improved method for extracting total RNA used to RT-PCR from microalage

以绿色巴夫藻（Pavlovaviridis）为实验材料，在比较了Trizol试剂法和异硫氰酸胍一步法这两种常用RNA提取方法的基础上，建立了一种十六烷基三乙基溴化铵（CTAB）和酸酚相结合的改良的CTAB-酸酚法。此方法利用高浓度的β-巯基乙醇来防止RNA提取过程中多酚的氧化，然后利用CTAB并结合乙醇和醋酸钾同时使用的方法来有效去除多糖类物质对RNA的干扰。经琼脂糖凝胶电泳检测，所提取的总RNA的28S、18S条带清晰明亮，无降解；其A260nm/A280nm值为1．968；200300nm全波长紫外扫描无多糖和蛋白吸收峰，表明提取的总RNA质量较好。对提取的总RNA直接进行反转录聚合酶链式反应（reverse transcription-polymerase chain reaction，RT—RCR），能够清楚地获得目的基因的cDNA片段，进一步证实了改良的CTAB-酸酚法是一种高质量提取微藻总RNA的有效方法。

Microalage contains lots of polysacchrides and polyphenols, which make it diihcult to extract to- tal RNA from Pavlova viridi. Compared with Trizol method and acid-guanidine-phenol-chloroform meth- od, an improved CTAB method which can use the high concentration β-Mercaptoethanol to eliminate the in terferences of polysacehrides and can use CTAB with both KAC and ethyl alcohol to eliminate the interfer- ences of polyphenols was constructed in this study. It was shown that 28S and ISS ribosomal RNA bands are clearly visible and the A260 nm/A280 nm ratio Of the isolated total RNA was 1. 968. Through scanning the isolated RNA by using the ultraviolet spectrophotometer, the result indicated that it is good to reduce polysacchrides and polyphenols in the sample. This isolated RNA was intact and had been proven suitable for further molecular applications by RT-PCR result. It was indicated that the method was appropriate for RNA extraction of microalage.