title: Quantitative stem-loop RT-PCR for detection of microRNAs.
abstract: Plant microRNAs (miRNAs) are a class of endogenous small RNAs that are essential for plant development and survival. They arise from larger precursor RNAs with a characteristic hairpin structure and regulate gene activity by targeting mRNA transcripts for cleavage or translational repression. Efficient and reliable detection and quantification of miRNA expression has become an essential step in understanding their specific roles. The expression levels of miRNAs can vary dramatically between samples and they often escape detection by conventional technologies such as cloning, northern hybridization and microarray analysis. The stem-loop RT-PCR method described here is designed to detect and quantify mature miRNAs in a fast, specific, accurate and reliable manner. First, a miRNA-specific stem-loop RT primer is hybridized to the miRNA and then reverse transcribed. Next, the RT product is amplified and monitored in real time using a miRNA-specific forward primer and the universal reverse primer. This method enables miRNA expression profiling from as little as 10 pg of total RNA and is suitable for high-throughput miRNA expression analysis.
authors.affiliation: The New Zealand Institute for Plant and Food Research, Mt. Albert Research Centre, Private Bag 92169, Auckland Mail Centre, Auckland 1142, New Zealand. Erika.Varkonyi-Gasic@plantandfood.co.nz
authors.forename: Erika
authors.initials: E
authors.lastname: Varkonyi-Gasic
authors.forename: Roger P
authors.initials: RP
authors.lastname: Hellens
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D055029:Inverted Repeat Sequences / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D010944:Plants / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D035683:MicroRNAs