Abstract
Quantitative real-time PCR (qRT-PCR) is a reliable method to determine and monitor microRNA (miRNA) expression profiles in different cells, tissues, and organisms. Although there are several different strategies in performing qRT-PCR to determine miRNA expression, all of them have two steps in common: reverse transcription for obtaining cDNA from mature miRNA sequencing and standard real-time PCR for amplification of cDNA. This chapter demonstrates the application of quantitative real-time PCR for determining miRNA expression profiles during mouse embryonic stem cell differentiation. In this method, a mature miRNA sequence is first reverse transcribed into a long cDNA with a 40–50 nt miRNA-specific stem-loop primer; then, a standard real-time PCR reaction is performed for determining miRNA expression using a forward miRNA-specific primer and a universal reverse primer.
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Acknowledgments
This work was partially supported by the East Carolina University New Faculty Research Startup Funds Program (to B.Z. and X.P.) and a Science and Engineering Grant from DuPont (to B.Z.).
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Pan, X., Murashov, A.K., Stellwag, E.J., Zhang, B. (2017). Using Quantitative Real-Time PCR to Detect MicroRNA Expression Profile During Embryonic Stem Cell Differentiation. In: Zhang, B. (eds) RNAi and Small Regulatory RNAs in Stem Cells. Methods in Molecular Biology, vol 1622. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7108-4_18
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DOI: https://doi.org/10.1007/978-1-4939-7108-4_18
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