title: Stem-Loop qRT-PCR for the Detection of Plant microRNAs.
abstract: Plant microRNAs (miRNAs) play important roles in the posttranscriptional regulation of protein-coding genes, and they are essential for a normal development and survival. Mature miRNAs are cleaved from larger precursor RNAs and are typically 21-22 nt long.The small size, the lack of a common feature like a poly(A) tail, 3' end-modifications, and presence of a precursor-all these factors affect the detection and hinder the quantification of miRNAs. The stem-loop qRT-PCR method described here is designed to detect and quantify mature miRNAs in a fast, specific, accurate, and reliable manner. Firstly, a miRNA-specific stem-loop RT primer is hybridized to miRNA and then reverse transcribed. Next, the RT product is amplified and monitored in real time using a miRNA-specific forward primer and a universal reverse primer. This method enables miRNA expression profiling from as little as 10 pg of total RNA, and it is suitable for a relatively high-throughput analysis of miRNA expression.
authors: The New Zealand Institute for Plant & Food Research Limited (Plant & Food Research) Mt Albert, Private Bag 92169, Auckland, 1142, New Zealand. erika.varkonyi-gasic@plantandfood.co.nz.
authors: Erika
authors: E
authors: Varkonyi-Gasic
mesh_terms: D017931:DNA Primers; D020869:Gene Expression Profiling / Q000379:methods; D006868:Hydrolysis; D055029:Inverted Repeat Sequences; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010944:Plants / Q000235:genetics; D018749:RNA, Plant; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D035683:MicroRNAs; D018749:RNA, Plant
keywords: SYBR Green; Stem-loop RT; UPL; miRNA; qPCR