Profiling MicroRNAs by Real-Time PCR
- 2.5k Downloads
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.
- 1.Kim, N., Han, J., and Siomi, M.C. (2009) Biogenesis of small RNAs in animals. Nature Reviews Molecular Cell Biology 10, 126–139.Google Scholar
- 2.Dieffenbach, C.W., Lowe, T.M., and Dveksler, D.S. (1993) General concepts for PCR primer design Genome Res. 3, S30–S37.Google Scholar
- 3.Raymond, C.K, Roberts, B.S., Garrett-Engele, P., Lim, L.P., and Johnson, J.M. (2005) RNA 11, 1737–1744.Google Scholar
- 10.Rozen, S. and Skaletsky, H.(2000) Primer3 on the WWW for general users and for biologist programmers. Bioinformatics Methods and Protocols in the series Methods in Molecular Biology. Humana Press, Totowa, NJ, 132, 365–386.Google Scholar
- 12.Griffiths-Jones, S. (2004) The microRNA Registry. Nucleic Acids Res. 32, Database issue D109–D111.Google Scholar
- 13.Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3, 34.1–34.11.Google Scholar
- 14.Andersen C.L., Jensen, J.L. and Ørentoft, T.F., (2004) Normalization of Real-Time Quantitative Reverse Transcription-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.PubMedCrossRefGoogle Scholar