迟钝爱德华氏菌RNA提取及痕量基因组DNA去除方法探究  

RNA Isolation from Edwardsiella tarda and Removal of Contaminating Genomic DNA in RNA Samples

作　　者：王克平[1] 叶江[1] 张惠展[1] 

机构地区：[1]华东理工大学生物工程学院、生物反应器工程国家重点实验室,上海200237

出　　处：《生物技术通报》2012年第6期179-182,共4页Biotechnology Bulletin

基　　金：生物反应器工程国家重点实验室重点项目配套子课题(2060204)

摘　　要：迟钝爱德华氏菌EIB202是一类细胞壁结构特殊的革兰氏阴性菌,高质量RNA提取相对较难。为了从转录组水平研究这类致病菌的致病机理,需要摸索有效的RNA提取及RNA样品中痕量基因组DNA去除方法。对常规RNA提取步骤进行改进,增加PBS清洗、反复冻融及较高浓度溶菌酶处理等步骤;另外,利用小体系基因组DNA去除系统,Mg2+与Mn2+协同激活DNase I去除RNA样品中基因组DNA污染。利用优化方法提取的RNA在质量及浓度(1 740 ng/μL)方面均有了显著改善,并建立了一套完全去除RNA样品中痕量基因组DNA污染的程序。The RNA isolation of Edwardsiella tarda EIB202, a Gram-negative bacterium with specific cell wall, is relatively difficult. It is necessary to explore the RNA isolation from Edwardsiella tarda and removal of contaminated genomic DNA in RNA samples for the purpose of researching virulence mechanism at the transcription level. On the basis of conventional RNA extraction method, an improvement method which added steps of washing with PBS, repeated freeze-thaw and processing with higher concentrations of lysozyme was used for the RNA isolation from Edwardsiella tarda. Furthermore, the method of using Mg^2＋ and Mn^2＋ in a microvolume digestion procedure was applied for removal of contaminated genomic DNA in RNA samples. High-quality RNA from Edwardsiella tarda was obtained using improved method. RNA purified by the removal method was used for RT-PCR and none band of DNA contamination could be observed in agarose gel.

关 键 词：迟钝爱德华氏菌 RNA 基因组 DNA DNASE I RT-PCR 

分 类 号：Q78[生物学—分子生物学]