Methodologies for high-throughput expression profiling of microRNAs.

MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression controlling central biological processes. These small, approx 22-nucleotide (nt)-long RNA molecules induce translational suppression when they are imperfectly matched to their target messenger RNA (mRNA) or direct mRNA cleavage when perfectly, or nearly perfectly, matched to their target. Direct roles in developmental processes have been described in a variety of species, and involvement in human diseases, such as cancer and diabetes, has been implied. These studies highlight the need to obtain detailed expression profiles of miRNAs in tissues, during development, and in disease. Their small size and the existence of miRNA families of related sequences pose critical problems in approaching expression analysis of miRNAs, especially using high-throughput approaches. All methodologies presented here address the special requirements for the analysis of miRNA expression using a variety of platforms, including cloning, microarrays, and microbeads. The different variables, as well as the different approaches, used by various laboratories are detailed and general recommendations are provided.