活性污泥高质量RNA 快速提取方法研究.pdfVIP

31 1 Vol. 31, No. 1 2010 1 Jan. , 2010 ENVIRONMENTAL SCIENCE RNA , , , , , , , , , * ( , 300050) : RNA , 5 RNA RNA RNA , TENP PBS , TRIzol DNA DNase I DNA , RNA. , RNA, RNA ( g 1696 g RNA) , 16S rRNA amoA RTPCR , , , RNA , ,TRFLPRNA , RNA . RNA , . : ;RNA ;TRFLP : X172 : A : 2010) Rapid Method to Extract HighQuality RNA fromActivated Sludge JIN Min, ZHAO Zuguo, QIU Zhigang, WANG Jingfeng, CHEN Zhaoli, SHEN Zhiqiang, LI Chao, WANG Xin wei, DONG Yan, LI Junwen ( Department of Environmental Health and Engineering, Institute of Hygiene and Environmental Medicine, Academy of Military Medical Sciences, Tianjin 300050, China) Abstract: An effective and fast RNA isolation method of activated sludge was established and five different methods were compared based on RNA yield, purity, integrity, RTPCR amplification of 16S rRNA genes and subsequent terminal restriction fragment length polymorphism ( TRFLP) analysis. That is, the precipitated activated sludge was washed with TENP and PBS buffer, followed by using lysozyme and TRIzol to direct lysis of microbial cells, chloroform to remove protein and most of the DNA from bacterial lysate, isopropanol to precipitate nucleic acid and DNase I to hydrolyze residual DNA. To further purify RNA , RNA purifying column was utilized. The results demonstrated that the extraction method, with the aid of TRIzol and RNA purification kit, can effectively extract highquality RNA . It not only means low degradability and high quantity, purity and diversity, but also the genes of 16S rRNA and amoA can be amplified by RTPCR. Compared with other methods, it showed great advantage of low cost and high efficienc