Journal of RNAi and Gene Silencing (August 2005), 1(1), 44-49

New Methods and Technologies

PCR-based expression analysis and identification of microRNAs

David P Lu †, Rebecca L Read †, David T Humphreys ‡, Fiona M Battah †, David I K Martin ‡ and John E J Rasko †§*

ABSTRACT

microRNAs (miRNAs) are small RNAs that regulate translation and hence control a variety of cellular processes in metazoans. The quantitation and identification of miRNAs has been hampered by their small size and low abundance. Here we describe two robust PCR-based assays of miRNA expression based on the original cloning strategy. The non-quantitative PCR method allows detection and identification of miRNAs and we utilise this method in the discovery of a new miRNA (miR-532) in retinoic acid differentiated P19 cells. The second and quantitative method (QM-RT-PCR) is simple and accurate, and uses commonly available technology. Of particular interest is the specificity of this PCR-based technology compared to hybridisation-based methods including arrays and northern blotting. Here we have shown that a single base pair mismatch in the priming sequence results in a two order of magnitude reduction in the amplification of let-7f. These streamlined methods will complement previously described methods and will facilitate analysis of miRNA expression in rare cell populations where the amount of RNA is limited.

KEYWORDS: microRNA, miR-532, RT-PCR, TaqMan, differentiation, hairpin RNA, let-7

† Gene and Stem Cell Therapy, Centenary Institute of Cancer Medicine and Cell Biology, University of Sydney, Locked Bag No 6, Newtown 2042, Australia.

‡ Victor Chang Cardiac Research Institute, Sydney, Australia.

§ Sydney Cancer Centre, Royal Prince Alfred Hospital, Sydney, Australia.

*Correspondence to: John E J Rasko, Email: j.rasko@centenary.usyd.edu.au, Tel: +612 95656156, Fax: +612 95656101

© Copyright David P Lu et al

(Received 19 May 2005; Revised 16 June 2005; Accepted 16 June 2005)