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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References
Basak I, Patil KS, Alves G et al (2016) MicroRNAs as neuroregulators, biomarkers and therapeutic agents in neurodegenerative diseases. Cell Mol Life Sci 73:811–827. https://doi.org/10.1007/s00018-015-2093-x
Bertoli G, Cava C, Castiglioni I (2016) MicroRNAs as biomarkers for diagnosis, prognosis and theranostics in prostate cancer. Int J Mol Sci 17:421. https://doi.org/10.3390/ijms17030421
Sastre B, Cañas JA, Rodrigo-Muñoz JM et al (2017) Novel modulators of asthma and allergy: exosomes and microRNAs. Front Immunol 8:826. https://doi.org/10.3389/fimmu.2017.00826
Bissels U, Wild S, Tomiuk S et al (2009) Absolute quantification of microRNAs by using a universal reference. RNA 15:2375–2384. https://doi.org/10.1261/rna.1754109
Bosson AD, Zamudio JR, Sharp PA (2014) Endogenous miRNA and target concentrations determine susceptibility to potential ceRNA competition. Mol Cell 56:347–359. https://doi.org/10.1016/j.molcel.2014.09.018
Chen C, Ridzon DA, Broomer AJ et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33:e179. https://doi.org/10.1093/nar/gni178
Afonina I, Zivarts M, Kutyavin I et al (1997) Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res 25:2657–2660
Kutyavin IV, Lukhtanov EA, Gamper HB et al (1997) Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization. Nucleic Acids Res 25:3718–3723
Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622. https://doi.org/10.1373/clinchem.2008.112797
Yang L, Wang S, Tang L et al (2014) Universal stem-loop primer method for screening and quantification of microRNA. PLoS One 9:e115293. https://doi.org/10.1371/journal.pone.0115293
Nolan T, Hands RE, Ogunkolade W et al (2006) SPUD: a quantitative PCR assay for the detection of inhibitors in nucleic acid preparations. Anal Biochem 351:308–310. https://doi.org/10.1016/j.ab.2006.01.051
Bustin SA, Benes V, Garson J et al (2013) The need for transparency and good practices in the qPCR literature. Nat Methods 10:1063–1067. https://doi.org/10.1038/nmeth.2697
Bustin SA (2014) The reproducibility of biomedical research: sleepers awake! Biomol Detect Quantif 2:35–42. https://doi.org/10.1016/j.bdq.2015.01.002
Vandesompele J, De Preter K, Pattyn F et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:research0034
Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250. https://doi.org/10.1158/0008-5472.CAN-04-0496
Pfaffl MW, Tichopad A, Prgomet C et al (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108
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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