一种广泛适用的RNA提取方法  

A RNA Isolation Method Suitable for a Wide Range of Materials

作　　者：淳俊[1] 郑彦峰[1] 王胜华[1] 陈放[1] 

机构地区：[1]四川大学生命科学学院,成都610064

出　　处：《生物化学与生物物理进展》2008年第5期591-597,共7页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(30270090);四川大学科研启动基金资助项目~~

摘　　要：分离提取高质量的RNA是基因表达、调控与基因工程等研究的基础,而RNase、多糖及多酚类物质严重干扰RNA的分离提取过程.现利用硅藻土对RNase的吸附性,结合PVP、高盐及乙二醇丁醚沉淀等处理,建立了一种广泛适用的RNA提取方法.在富含多糖的玉米胚乳,富含RNase的动物肝脏,多酚多油脂的银杏、麻疯树以及木霉、酵母等10多种RNA提取困难的动、植物与微生物材料中都提取出完整性好,得率高的RNA.RT-PCR实验表明,提取的RNA能够用于后续的分子生物学研究.硅藻土-苯酚法提取RNA的得率是异硫氰酸胍法的3倍多.此外,将分离提取的总RNA经过LiCl与PEG8000加NaCl沉淀步骤有效地去除了大片段RNA,以水稻Osa-mir-156的成熟序列设计特异引物做茎环RT-PCR,结果证明,富集得到的小RNA可以用于miRNA克隆等后续实验.High levels of RNase, polysaccharides and polyphenol compounds make isolation of high quality RNA difficult. Thus it is presented an effective RNA extraction method based on the nuclease adsorbent macaloid, poly vinyl pyrrolidone, and high concentration of KAc and ethylene glycol monobutyl ether, which has successfully extracted high-quality RNA from many materials difficult to RNA isolation, such as RNase-rich rabbit liver, plant and microbial tissues rich in polysaccharides, lipids and polyphenol compounds. This method was found to be better than the ones in common use-Trizol and Guanidinium isothiocyanate, the yield of which was at least three time higher. Furtherly, small RNA was enriched from total RNA sample from rice seedling through by repeat deposit which deals with high concentration of LiCl, PEG8000 and NaCI. The small RNA gained was confirmed to be used for following molecular biological research by RT-PCR with the primers designed on osa-mir-156 sequence from rice miRNA.

关 键 词：硅藻土 RNA提取 小RNA 乙二醇丁醚 

分 类 号：Q781[生物学—分子生物学]