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miRNA Detection by Stem-Loop RT-qPCR in Studying microRNA Biogenesis and microRNA Responsiveness to Abiotic Stresses

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Plant MicroRNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1932))

Abstract

This chapter is devoted to a PCR-based method for analyzing the expression level of mature miRNAs which utilizes the TaqMan® technology. Stem-loop RT-qPCR requires preparation of separate cDNA templates for each analyzed miRNA as reverse transcription occurs in the presence of a miRNA-specific stem-loop reverse primer. In quantitative analysis, SYBR® Green is not used but the more sensitive TaqMan® probe that on 5′ end contains a covalently attached fluorophore and on 3′ quencher. When quencher and fluorophore are spatially separated due to nucleolytic DNA polymerase activity, the signal is released and quantified. This section provides a detailed and comprehensive protocol allowing for the successful analysis of mature miRNA levels in analyzed sample. Reverse transcription combined with classic real-time PCR as well as ddPCR™ (Droplet Digital™ PCR) will be presented.

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Acknowledgment

The project is supported by the National Science Centre, Poland based on the decisions number DEC-2013/11/B/NZ9/01761, UMO-2015/19/N/NZ9/00218, 2013/10/A/NZ1/00557, UMO-2016/23/N/NZ1/00005, UMO-2016/21/B/NZ9/00550, UMO-2016/23/B/NZ9/00857 and UMO-2016/23/B/NZ9/00862, by the Foundation for Polish Science (grants START 2017 to Agata S. and Katarzyna K.), and by KNOW RNA Research Centre in Poznań (No. 01/KNOW2/2014).

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Authors and Affiliations

  1. Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poznań, Poland

    Aleksandra Smoczynska, Pawel Sega, Agata Stepien, Katarzyna Knop, Artur Jarmolowski, Andrzej Pacak & Zofia Szweykowska-Kulinska

Authors
  1. Aleksandra Smoczynska
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  2. Pawel Sega
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  3. Agata Stepien
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  4. Katarzyna Knop
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  5. Artur Jarmolowski
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  6. Andrzej Pacak
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  7. Zofia Szweykowska-Kulinska
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Corresponding authors

Correspondence to Andrzej Pacak or Zofia Szweykowska-Kulinska .

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Editors and Affiliations

  1. Unidad de Genómica Avanzada, Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Irapuato, Guanajuato, Mexico

    Stefan de Folter

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Smoczynska, A. et al. (2019). miRNA Detection by Stem-Loop RT-qPCR in Studying microRNA Biogenesis and microRNA Responsiveness to Abiotic Stresses. In: de Folter, S. (eds) Plant MicroRNAs. Methods in Molecular Biology, vol 1932. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9042-9_10

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  • DOI: https://doi.org/10.1007/978-1-4939-9042-9_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9041-2

  • Online ISBN: 978-1-4939-9042-9

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