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