Abstract
Probe-based quantitative PCR (qPCR) is a commonly used tool in the realm of real-time qPCR experiments since it is one of the most sensitive detection methods allowing an accurate and reproducible analysis. It uses real-time fluorescence from a fluorescently labeled probe that specifically targets the desired PCR product to measure DNA amplification at each cycle of the PCR. Coupled to a proper reverse transcription step, probe-based qPCR can be efficiently used for the analysis of the expression of difficult targets such as miRNAs. In this chapter, we describe the TaqMan® advanced miRNA assay in which, owing to a poly(A)-tailing step, the reverse transcription is advantageously performed at once for all the miRNAs in a given sample, and, coupled to the ligation of a 5′ universal adapter, allows for a supplementary pre-qPCR amplification step increasing the sensitivity of the assay. Along this protocol, we also provide our general guidelines and advices to perform a reliable and successful quantitative analysis.
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Acknowledgments
This work was supported by a public grant overseen by the French National Research Agency (ANR) as part of the second “Investissements d’Avenir” program FIGHT-HF (reference: ANR-15-RHU-0004).
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Vautrot, V., Behm-Ansmant, I. (2020). Enhanced Probe-Based RT-qPCR Quantification of MicroRNAs Using Poly(A) Tailing and 5′ Adaptor Ligation. In: Biassoni, R., Raso, A. (eds) Quantitative Real-Time PCR. Methods in Molecular Biology, vol 2065. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9833-3_4
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DOI: https://doi.org/10.1007/978-1-4939-9833-3_4
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