Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available

We review different methodologies to estimate the expression levels of microRNAs (miRNAs) using real-time quantitative PCR (qPCR). As miRNA analysis is a fast changing research field, we have introduced novel technological approaches and compared them to existing qPCR profiling methodologies. qPCR also remains the method of choice for validating results obtained from whole-genome screening (e.g. with microarray). In contrast to presenting a stepwise description of different platforms, we discuss expression profiling of mature miRNAs by qPCR in four sequential sections: (1) cDNA synthesis; (2) primer design; (3) detection of amplified products; and (4) data normalization. We address technical challenges associated with each of these and outline possible solutions.