Chapter title |
Using Quantitative Real-Time PCR to Detect MicroRNA Expression Profile During Embryonic Stem Cell Differentiation
|
---|---|
Chapter number | 18 |
Book title |
RNAi and Small Regulatory RNAs in Stem Cells
|
Published in |
Methods in molecular biology, July 2017
|
DOI | 10.1007/978-1-4939-7108-4_18 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7106-0, 978-1-4939-7108-4
|
Authors |
Pan, Xiaoping, Murashov, Alexander K., Stellwag, Edmund J., Zhang, Baohong, Xiaoping Pan, Alexander K. Murashov, Edmund J. Stellwag, Baohong Zhang |
Editors |
Baohong Zhang |
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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