title: Amplification of unknown RNAs and RNA mixtures based on unique restriction enzyme cleavage in vitro.
abstract: 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.
authors.affiliation: Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, China.
authors.forename: Fangyi
authors.initials: F
authors.lastname: Yang
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.forename: Yajun
authors.initials: Y
authors.lastname: Ji
authors.forename: Hao
authors.initials: H
authors.lastname: Cheng
authors.forename: Junting
authors.initials: J
authors.lastname: Wan
authors.forename: Zhiyu
authors.initials: Z
authors.lastname: Xiao
authors.forename: Guochun
authors.initials: G
authors.lastname: Zhou
mesh_terms: D001483:Base Sequence; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D011130:RNA Ligase (ATP) / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D014764:Viral Proteins; D012313:RNA; D004262:DNA Restriction Enzymes; D011088:DNA Ligases; D011130:RNA Ligase (ATP); C477285:bacteriophage T4 RNA ligase 1