alexa Abstract | PCR-based expression analysis and identification of microRNAs

Journal of RNAi and Gene Silencing
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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.

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Author(s): David P Lu Rebecca L Read David T Humphreys Fiona M Battah David I K Martin and John E J Rasko

Keywords

microRNA, miR-532, RT-PCR, TaqMan, differentiation, hairpin RNA, let-7, RNAi, Gene Silencing

 
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