Abstract
Stem-loop qRT-PCR is one of the most commonly used real-time PCR approach to quantify small non-coding RNAs such as microRNAs. The quantification method is divided in two steps. First, RNA is reverse-transcribed using a specific stem-loop primer, and the resulting RT product is subsequently used as a template for quantitative real-time PCR. This fast and simple method provides quantitative data with high sensitivity and specificity to study miRNAs and their functions.
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References
Kwok S, Higuchi R (1989) Avoiding false positives with PCR. Nature 339(6221):237–238. doi:10.1038/339237a0
Mullis KB, Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335–350. doi:10.1016/0076-6879(87)55023-6 [pii]
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408. doi:10.1006/meth.2001.1262, S1046-2023(01)91262-9 [pii]
Galiveti CR, Rozhdestvensky TS, Brosius J, Lehrach H, Konthur Z (2010) Application of housekeeping npcRNAs for quantitative expression analysis of human transcriptome by real-time PCR. RNA 16(2):450–461. doi:10.1261/rna.1755810, rna.1755810 [pii]
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(6):R64. doi:10.1186/gb-2009-10-6-r64, gb-2009-10-6-r64 [pii]
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 26(6):509–515
Wei CH, Kao HY (2011) Cross-species gene normalization by species inference. BMC Bioinform 12(Suppl 8):S5. doi:10.1186/1471-2105-12-S8-S5, 1471-2105-12-S8-S5 [pii]
Andersen CL, Jensen JL, Ørntoft T (2004) Normalization of real-time quantitative RT-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
Acknowledgment
This work was supported by the Centre National pour la Recherche Scientifique, the Université de Lorraine, the Région Lorraine and the Ligue nationale pour la recherche contre le cancer (comités 54 et 57).
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Salone, V., Rederstorff, M. (2015). Stem-Loop RT-PCR 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_10
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DOI: https://doi.org/10.1007/978-1-4939-2547-6_10
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2546-9
Online ISBN: 978-1-4939-2547-6
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