title: Poly(T) adaptor RT-PCR.
abstract: Reverse transcription PCR (RT-PCR) is one of the most important techniques for analyzing RNA abundance. MicroRNAs (miRNAs) are a group of 20- to 24-nucleotide regulatory small RNAs which play an important role in plants and animals. However, the small size of miRNAs makes them difficult to be detected and quantified by conventional RT-PCR techniques. Here, we describe a poly(T) adaptor RT-PCR method specifically designed for quantifying miRNAs. In this method, total RNAs, including miRNAs, are extended by a poly(A) tailing reaction using poly(A) polymerase and ATP. The miRNA with a poly(A) tail is converted into cDNA through reverse transcription primed by a poly(T) adaptor, and then PCR-amplified using a miRNA-specific forward primer and a universal poly(T) adaptor reverse primer. The RT-PCR amplification can be monitored by real-time detection or by end-point detection for quantifying the miRNA transcript level. The PCR amplicons can be sequenced for validating the expression of the specific miRNA gene.
authors.affiliation: Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Shi
authors.forename: Ying-Husan
authors.initials: YH
authors.lastname: Sun
authors.forename: Xing-Hai
authors.initials: XH
authors.lastname: Zhang
authors.forename: Vincent L
authors.initials: VL
authors.lastname: Chiang
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D035683:MicroRNAs / Q000032:analysis* / Q000737:chemistry; D011071:Poly T; D012313:RNA / Q000032:analysis / Q000302:isolation & purification; D018749:RNA, Plant / Q000032:analysis / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D017931:DNA Primers; D035683:MicroRNAs; D018749:RNA, Plant; D011071:Poly T; D012313:RNA