In vitro analysis of microRNA processing using recombinant Dicer and cytoplasmic extracts of HeLa cells

MicroRNAs (miRNAs) constitute a novel class of short, non-coding RNAs that play crucial roles as silencers of gene expression during biological processes such as development, growth control, cell death, and differentiation. To ensure correct function, the expression of miRNAs must be tightly regulated, a process that is believed to take place at the promoter level. Regulation of the miRNA processing cascade has only recently been shown to play an important role as well and we envision the discovery of additional factors affecting or modulating miRNA processing and ultimately miRNA expression. The biochemical analysis of such factors requires a robust assay to monitor miRNA processing. Here, we discuss protocols and techniques that were used to investigate how the expression of brain-specific miRNA miR-138 is controlled at the level of precursor-miRNA (pre-miRNA) processing.