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Plant Epigenetics pp 163–175Cite as

Stem-Loop qRT-PCR for the Detection of Plant microRNAs

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1456))

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.

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Author information

Authors and Affiliations

  1. The New Zealand Institute for Plant & Food Research Limited (Plant & Food Research) Mt Albert, Private Bag 92169, Auckland, 1142, New Zealand

    Erika Varkonyi-Gasic

Authors
  1. Erika Varkonyi-Gasic
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Correspondence to Erika Varkonyi-Gasic .

Editor information

Editors and Affiliations

  1. Department of Biological Sciences, University of Lethbridge, Lethbridge, Alberta, Canada

    Igor Kovalchuk

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Varkonyi-Gasic, E. (2017). Stem-Loop qRT-PCR for the Detection of Plant microRNAs. In: Kovalchuk, I. (eds) Plant Epigenetics. Methods in Molecular Biology, vol 1456. Humana Press, Boston, MA. https://doi.org/10.1007/978-1-4899-7708-3_13

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  • DOI: https://doi.org/10.1007/978-1-4899-7708-3_13

  • Published:

  • Publisher Name: Humana Press, Boston, MA

  • Print ISBN: 978-1-4899-7706-9

  • Online ISBN: 978-1-4899-7708-3

  • eBook Packages: Springer Protocols

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