嫩芽总RNA的提取与纯化

Extraction and purification of total RNA from young buds of Nai(Prunus salicina Lindl.var.cordata)

以嫩芽为材料,对Trizol法进行改进提取总RNA,试验结果表明,加入10%PVPP与材料共研磨,并在Trizol提取液中加入1%β-巯基乙醇,可以完全排除酚类物质、色素等杂质的干扰;采用在匀浆液中加入终浓度为20%乙醇沉淀多糖、在裂解液抽提的上相中加入终浓度为3mol/LLiCl沉淀RNA、在RNA的粗提液中加入适量的3mol/LNaAc(pH5.2)和低浓度的无水乙醇沉淀多糖等方法相结合,可以有效地去除多糖的干扰,获得纯净、完整的RNA样品,用它为模板进行RT-PCR反应,获得PPO基因保守区约600～800bp的cDNA条带;而单独采用在匀浆液中加入终浓度为20%乙醇沉淀多糖的方法,不能将样品中的多糖去除干净,这些残留的多糖会抑制逆转录酶的活性,导致逆转录反应的失败。

:Total RNA of Nai's young buds was extracted with modified Trizol method.The results showed that the impurities such as polyphenolics, pigments etc were completely removed by adding 10% Poly Vinyl Poly Pyrrilodone to the grinded materials under liquid nitrogen in mortar and adding 1% β-mercaptoethanol to Trizol extracting buffer ;that polysaccharides were effectively removed by combination adding absolute ethanol to 20% final concentration into homogenization solution to precipitate polysaccharides, adding LiCl to 3 mol/L final concentration into aqueous phase after extracting with chloroform to precipitate RNA with adding proper volume 3 mol/L NaAc(pH 5.2) and low concentration of absolute ethanol into rough solution of extracted RNA to precipitate polysaccharides in order to obtain pure and intact RNA samples for templates of reverse-transcription and polymerase chain reaction and one 600-800bp cDNA special band of conserve zone in PPO gene was amplified; that polysaccharides still remained in RNA samples alone by adding absolute ethanol to 20% final concentration into homogenization solution to precipitate polysaccharides, the remaining polysaccharides inhibited activity of reverse-transcriptase and resulted in failure of reverse-transcription reaction.