title: PCR-based expression analysis and identification of microRNAs.
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.
authors.affiliation: Gene and Stem Cell Therapy, Centenary Institute of Cancer Medicine and Cell Biology, University of Sydney, Locked Bag No 6, Newtown 2042, Australia.
authors.forename: David P
authors.initials: DP
authors.lastname: Lu
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Read
authors.forename: David T
authors.initials: DT
authors.lastname: Humphreys
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Battah
authors.forename: David I K
authors.initials: DI
authors.lastname: Martin
authors.forename: John E J
authors.initials: JE
authors.lastname: Rasko