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RT-qPCR-Based Quantification of Small Non-Coding RNAs

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Small Non-Coding RNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1296))

Abstract

MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively regulate messenger RNA (mRNA) translation into protein. MiRNAs play a key role in gene expression regulation, and their involvement in disease biology is well documented. This has fueled the development of numerous tools for the quantification of miRNA expression levels. These tools are based on three technologies: (microarray) probe hybridization, RNA sequencing, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). In this chapter, we describe a quantification system based on RT-qPCR technology, which is currently considered as the most sensitive, flexible, and accurate method for quantification of not only miRNA but also RNA expression in general. To this purpose, we have divided the protocol in three sections: reverse transcription (RT) reaction, optional preamplification (PA), and finally qPCR. Three quality-control (QC) steps are implemented in this workflow for assessment of RNA extraction efficiency, sample purity (e.g., absence of inhibitors), and inter-run variations, by examining the detection level of different spike-in synthetic miRNAs. We conclude by demonstrating raw data preprocessing and normalization using expression data obtained from high-throughput miRNA profiling of human RNA samples.

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References

  1. Krol J, Loedige I, Filipowicz W (2010) The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet 11:597–610

    CAS  PubMed  Google Scholar 

  2. Kozomara A, Griffiths-Jones S (2013) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42:D68–D73

    Article  PubMed Central  PubMed  Google Scholar 

  3. Berezikov E (2011) Evolution of microRNA diversity and regulation in animals. Nat Rev Genet 12:846–860

    Article  CAS  PubMed  Google Scholar 

  4. Soifer HS, Rossi JJ, Sætrom P (2007) MicroRNAs in disease and potential therapeutic applications. Mol Ther 15:2070–2079

    Article  CAS  PubMed  Google Scholar 

  5. Lesko LJ (2007) Personalized medicine: elusive dream or imminent reality? Clin Pharmacol Ther 81:807–816

    Article  CAS  PubMed  Google Scholar 

  6. Schwarzenbach H, Hoon DSB, Pantel K (2011) Cell-free nucleic acids as biomarkers in cancer patients. Nat Cell Biol 11:426–437

    CAS  Google Scholar 

  7. Mestdagh P, Hartmann N, Baeriswyl L, Andreasen D, Bernard N, Chen C, Cheo D, D’Andrade P, DeMayo M, Dennis L, Derveaux S, Feng Y, Fulmer SS, Gerstmayer B, Gouffon J, Grimley C, Lader E, Lee KY, Luo S, Mouritzen P, Narayanan A, Patel S, Peiffer S, Rüberg S, Schroth G, Schuster D, Shaffer JM, Shelton EJ, Silveria S, Ulmanella U, Veeramachaneni V, Staedtler F, Peters T, Guettouche T, Wong L, Vandesompele J (2014) Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study. Nat Methods 11:809–815

    Article  CAS  PubMed  Google Scholar 

  8. Mestdagh P, Van Vlierberghe P, De Weer A, Muth D, Westermann F, Speleman F, Vandesompele J (2009) A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol 10:R64

    Article  PubMed Central  PubMed  Google Scholar 

  9. D’haene B, Mestdagh P, Hellemans J, Vandesompele J (2012) MiRNA expression profiling: from reference genes to global mean normalization. Methods Mol Biol 822:261–272

    Article  PubMed  Google Scholar 

  10. Ståhlberg A, Håkansson J, Xian X, Semb H, Kubista M (2004) Properties of the reverse transcription reaction in mRNA quantification. Clin Chem 50:509–515

    Article  PubMed  Google Scholar 

  11. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:R19

    Article  PubMed Central  PubMed  Google Scholar 

  12. Aranda R, Dineen SM, Craig RL, Guerrieri RA, Robertson JM (2009) Comparison and evaluation of RNA quantification methods. Anal Biochem 387:122–127

    Article  CAS  PubMed  Google Scholar 

  13. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3 research0034.1–1333

    Google Scholar 

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Acknowledgment

Evaluation of this workflow was supported by Fournier-Majoie Foundation.

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Correspondence to Jo Vandesompele .

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Zeka, F., Mestdagh, P., Vandesompele, J. (2015). RT-qPCR-Based Quantification of Small Non-Coding RNAs. In: Rederstorff, M. (eds) Small Non-Coding RNAs. Methods in Molecular Biology, vol 1296. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2547-6_9

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  • DOI: https://doi.org/10.1007/978-1-4939-2547-6_9

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2546-9

  • Online ISBN: 978-1-4939-2547-6

  • eBook Packages: Springer Protocols

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