Profiling MicroRNAs by Real-Time PCR
A variety of physiological processes are associated with changes in microRNA (miRNA) expression. Analysis of miRNA has been applied to study normal physiology as well as diseased states including cancer. One major challenge in miRNA research is to accurately and practically determine the expression level of miRNAs in various experimental systems. Many genome-wide miRNA expression profiling studies have relied on microarrays technology, and frequently differentially expressed miRNAs have subsequently been confirmed with real-time quantitative PCR studies. Here, we describe how different primer strategies for first-strand cDNA synthesis and PCR amplification can affect measurements of miRNA expression levels. Overcoming the small nature of miRNAs is a difficult task as the short sequence available does not allow for designing primers using standard PCR primer design guidelines. Finally, we demonstrate how to determine differentially expressed miRNAs using a locked nucleic acid-based real-time PCR approach.
Key wordsQuantitative real-time PCR Reverse transcription Amplification efficiency Quantification cycle SYBR Green I detection Primer design strategies Melting temperature
We thank Ms. Mette Carlsen Mohr and Ms. Madeline Ek for their skilled technical assistance. We also thank Rolf Søkilde for purification of the total RNA preparations. Finally, we thank Liselotte Kahns for the development of the endogenous control assays.
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