Plant Epigenetics

Volume 631 of the series Methods in Molecular Biology™ pp 109-122


qRT-PCR of Small RNAs

  • Erika Varkonyi-GasicAffiliated withHortResearch, Mt Albert Research Centre Email author 
  • , Roger P. HellensAffiliated withHortResearch, Mt Albert Research Centre

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Plant small RNAs are a class of 19- to 25-nucleotide (nt) RNA molecules that are essential for genome stability, development and differentiation, disease, cellular communication, signaling, and adaptive responses to biotic and abiotic stress. Small RNAs comprise two major RNA classes, short interfering RNAs (siRNAs) and microRNAs (miRNAs). Efficient and reliable detection and quantification of small RNA expression has become an essential step in understanding their roles in specific cells and tissues. Here we provide protocols for the detection of miRNAs by stem-loop RT-PCR. This method enables fast and reliable miRNA expression profiling from as little as 20 pg of total RNA extracted from plant tissue and is suitable for high-throughput miRNA expression analysis. In addition, this method can be used to detect other classes of small RNAs, provided the sequence is known and their GC contents are similar to those specific for miRNAs.

Key words

Small RNA miRNA RT Stem-loop RT qPCR SYBR Green I assay UPL probe assay