活性污泥高质量总RNA提取的一种有效方法

AN EFFECTIVE METHOD FOR EXTRACTION OF HIGH QUALITY RNA FROM ACTIVATED SLUDGE

提取高质量的RNA是在基因表达水平上研究废水生物处理过程活性污泥中微生物菌群变化及关键调控基因表达规律的必要条件,但由于活性污泥富含腐殖质与RNA酶,导致常规提取方法难以获得高质量的RNA.本研究通过反复探索与试验,得到了一种经济而快捷的从活性污泥中提取高质量RNA的方法,即在PBS洗涤沉淀污泥的基础上,用含PVP和巯基乙醇的CTAB(十六烷基溴化三甲胺)裂解液裂解活性污泥细胞,并与细胞中的核酸形成核酸-CTAB复合体,然后用酚-氯仿-异戊醇去除细胞裂解液中的蛋白,最后采用LiCl选择性沉淀其中的RNA.经核酸蛋白测定仪和1%变性琼脂糖凝胶电泳检测表明,用此方法提取的RNA均一性和完整性好,并且具有反转录活性.

Isolation of high-quality RNA from activated sludge is a prerequisite to study the diversity of microbial community and the expression pattern of key controlling genes at the gene expression level during the wastewater treatment process. Activated sludge is rich in humic substances and RNase which may cause low yield and degradation of RNA during the nucleic acid extraction with the conventional method. In this work, a more efficient method of RNA extraction was established from activated sludge. That is, the precipated activated sludge was washed with PBS buffer, followed by the direct lysis of microbial cells and inacti- vation of nucleases with the CTAB lysis buffer consisting of PVP and j3-mercaptoethanol, then by phenol-chloroform-isoamyl alcohol extraction and LiC1 precipation. The yielded total RNA by this method was of not only integrality and high purity but also simple manipulation and cheapness. Moreover, the extracted RNA was of reverse transcript activity.