Monitoring microRNA expression during embryonic stem-cell differentiation using quantitative real-time PCR (qRT-PCR).
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 for performing qRT-PCR to determine miRNA expression, all of them have two steps in common: reverse transcription for obtaining cDNA from mature miRNA sequence and standard real-time PCR for amplification of cDNA. This chapter demonstrates the application of TaqMan-based 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- to 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, a universal reverse primer, and FAM dye-labeled TaqMan probes.
Pan
Xiaoping
X
Department of Biology, East Carolina University, Greenville, NC, USA.
Murashov
Alexander K
AK
Stellwag
Edmund J
EJ
Zhang
Baohong
B
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D035683:MicroRNAs