Tiny giants of gene regulation: experimental strategies for microRNA functional studies.

The discovery over two decades ago of short regulatory microRNAs (miRNAs) has led to the inception of a vast biomedical research field dedicated to understanding these powerful orchestrators of gene expression. Here we aim to provide a comprehensive overview of the methods and techniques underpinning the experimental pipeline employed for exploratory miRNA studies in animals. Some of the greatest challenges in this field have been uncovering the identity of miRNA-target interactions and deciphering their significance with regard to particular physiological or pathological processes. These endeavors relied almost exclusively on the development of powerful research tools encompassing novel bioinformatics pipelines, high-throughput target identification platforms, and functional target validation methodologies. Thus, in an unparalleled manner, the biomedical technology revolution unceasingly enhanced and refined our ability to dissect miRNA regulatory networks and understand their roles in vivo in the context of cells and organisms. Recurring motifs of target recognition have led to the creation of a large number of multifactorial bioinformatics analysis platforms, which have proved instrumental in guiding experimental miRNA studies. Subsequently, the need for discovery of miRNA-target binding events in vivo drove the emergence of a slew of high-throughput multiplex strategies, which now provide a viable prospect for elucidating genome-wide miRNA-target binding maps in a variety of cell types and tissues. Finally, deciphering the functional relevance of miRNA post-transcriptional gene silencing under physiological conditions, prompted the evolution of a host of technologies enabling systemic manipulation of miRNA homeostasis as well as high-precision interference with their direct, endogenous targets. For further resources related to this article, please visit the WIREs website.