一种改良的热酚法高效快速提取酿酒酵母总RNA  

A Modified Method for the Efficient and Fast Extraction of Total RNA from Saccharomyces cerevisiae with Hot-Phenol

作　　者：李维维[1,2] 顿宝庆[2] 王智[2] 曲娟娟[1] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]中国农业科学院作物科学研究所中国农业科学院生物质能源研究中心,北京100081

出　　处：《生物技术通报》2012年第12期163-166,共4页Biotechnology Bulletin

基　　金：中国农业科学院作物科学研究所中央级公益性科研院所基本科研业务费专项资助项目(2060302-13)

摘　　要：以酿酒酵母单倍体CE N.PK1与双倍体CEN.PK2为研究对象,利用改良热酚法,快速高效地提取酿酒酵母总RNA。结果显示应用该方法提取的酿酒酵母CEN.PK1和CEN.PK2的总RNA经分光光度计测定浓度为7.63μg/μL和3.77μg/μL,OD_(260/280)分别为2.10和2.04,OD_(260/230)分别为2.32和2.24,浓度与纯度均能达到后续分子生物学试验的要求。经PCR与荧光定量PCR检测,该方法提取的RNA无DNA污染,可作为PCR反应的模板,与Trizol方法提取RNA相比,该方法不仅浓度高并且可将提取时间由常规的2.5 h缩短为1.5 h,具有操作简单、高质、高效等优点,经多次试验证明,此方法同样适用于酿酒酵母总RNA的大量提取试验,有较高的实用价值。To research an efficient method for total RNA extraction from Saccharomyces cerevisiae, haploid CEN.PKland diploid CEN. PK2 as the object of study was used to study the new modified hot-acid-phenol method. The total RNA concentration of CEN.PK1 and CEN.PK2 was 7.63 mg/mL and 3.77 mg/mL, OD260/280 was 2.10 and 2.04 and OD2ar230 was 2.32 and 2.24, using the modified method by spectrophotometer determination. It is proved that the RNA was fit for the requirements of follow-up experiments in molecular biology by PCR and fluorescence quantitative PCR detection without DNA contamination compared with the Trizol method. The extraction time 1.5 hours is shorter than that of conventional method 2.5 hours used and the modified method has the advantages of simple operation, high efficiency and high quality. Repeated experiments proved that this method can also be used to extract the total RNA amount of Saccharomyces cerevisiae and can be used for practical applications.

关 键 词：RNA分离 酿酒酵母 热酚法 CDNA 荧光定量PCR 

分 类 号：Q78[生物学—分子生物学]