Amplification of unknown RNAs and RNA mixtures based on unique restriction enzyme cleavage in vitro  

作　　者：Fangyi Yang Jie Wang Yajun Ji Hao Cheng Junting Wan Zhiyu Xiao Guochun Zhou 

机构地区：[1]Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China [2]Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, China [3]Department of Oncology, The First People's Hospital of Lianyungang, Lianyungang 222002, China

出　　处：《Acta Biochimica et Biophysica Sinica》2010年第12期873-882,共10页生物化学与生物物理学报（英文版）

摘　　要：Small RNAs, generally expressed at low levels, are difficult to reach usable levels from limited material. In this study, we have developed a novel method to amplify target RNA. The amplification procedure was carried out by sequential RT-PCR, effective separation, restriction enzymatic cleavage of cDNA strand, and run-off transcription in vitro of target RNA from its cDNA. Introduction of a unique stem-loop linker into cDNA strand is the key step to form a unique restriction enzyme recognition sequence that is not in cDNA sequence of target RNA. This method can be used to amplify RNA samples from various origins and has many advantages in amplifying unknown small RNAs and small RNA mixtures. The amplified RNA has the full sequence of original RNA except for an extra 5′ G and an additional 3 A or C. The method worked well for amplifications of a microRNA, a piwi interacting RNA and two small RNA mixtures.Small RNAs, generally expressed at low levels, are difficult to reach usable levels from limited material. In this study, we have developed a novel method to amplify target RNA. The amplification procedure was carried out by sequential RT-PCR, effective separation, restriction enzymatic cleavage of cDNA strand, and run-off transcription in vitro of target RNA from its cDNA. Introduction of a unique stem-loop linker into cDNA strand is the key step to form a unique restriction enzyme recognition sequence that is not in cDNA sequence of target RNA. This method can be used to amplify RNA samples from various origins and has many advantages in amplifying unknown small RNAs and small RNA mixtures. The amplified RNA has the full sequence of original RNA except for an extra 5′ G and an additional 3 A or C. The method worked well for amplifications of a microRNA, a piwi interacting RNA and two small RNA mixtures.

关 键 词：RNA amplification stem-loop linker unknown RNA RNA mixture 

分 类 号：Q52[生物学—生物化学] Q78