MicroRNA Profiling

In 1993, R.C. Lee of Harvard University first described miRNA-mediated silencing in C. elegans, and since, these molecules have been more clearly defined as single-stranded RNA molecules, 19-25 nucleotides in length, that are generated from endogenous hairpin transcripts. MicroRNAs (miRNAs) serve as guides in post-transcriptional gene silencing by complimentary base pairing with target mRNAs, resulting in mRNA cleavage or translational repression. As a result, miRNAs enable regulation of complex biological pathways such as those associated with developmental processes, haematopoietic cell differentiation, apoptosis, and cell proliferation. Interestingly, it now appears that miRNAs may actually form complex regulatory networks with target mRNAs, as a single miRNA may be responsible for the regulation of several different targets, or conversly, several miRNAs may cooperatively regulate a single mRNA target. To date, there have been approximately 4300 precursor miRNAs found in virtually all species—animals, plants, and viruses—of which ~475 are human miRNAs. Research suggests that as many as one-third of all human genes may be miRNA regulated, many of which are involved in cancer and other disease regulation.

Due to their involvement in gene regulation, miRNAs have received significant attention with respect to their role in cancer, disease, and stem cell differentiation. Traditional characterization of miRNA follows small RNA identification by cDNA cloning. Expression of miRNAs is typically confirmed by hybridization to a size-fractionated RNA sample, usually achieved by Nothern blot analysis. Alternative methods for miRNA detection and confirmation include reverse transcription PCR (RT-PCR), primer extension analysis, RNase protection assays, and microarray analysis. Typically, even when miRNAs are identified using these alternative methods, Northern blot analysis follows as it enables the confirmation of both the hairpin precursor (~70 nt) and mature miRNA (~22 nt) forms. Global gene expression profiling of miRNAs using microarrays provides high-throughput information on miRNA involvement in disease progression and developmental changes, while offering an alternative to some of the time- and labor-intensive techniques previously described.