Advances in microRNA experimental approaches to study physiological regulation of gene products implicated in CNS...

The central nervous system (CNS) is a remarkably complex organ system, requiring an equally complex network of molecular pathways controlling the multitude of diverse, cellular activities. Gene expression is a critical node at which regulatory control of molecular networks is implemented. As such, elucidating the various mechanisms employed in the physiological regulation of gene expression in the CNS is important both for establishing a reference for comparison to the diseased state and for expanding the set of validated drug targets available for disease intervention. MicroRNAs (miRNAs) are an abundant class of small RNA that mediates potent inhibitory effects on global gene expression. Recent advances have been made in methods employed to study the contribution of these miRNAs to gene expression. Here we review these latest advances and present a methodological workflow from the perspective of an investigator studying the physiological regulation of a gene of interest. We discuss methods for identifying putative miRNA target sites in a transcript of interest, strategies for validating predicted target sites, assays for detecting miRNA expression, and approaches for disrupting endogenous miRNA function. We consider both advantages and limitations, highlighting certain caveats that inform the suitability of a given method for a specific application. Through careful implementation of the appropriate methodologies discussed herein, we are optimistic that important discoveries related to miRNA participation in CNS physiology and dysfunction are on the horizon.