Identify MicroRNA Targets Using AGO2-CLASH (Cross-linking, Ligation, and Sequencing of Hybrids) and AGO2-CLIP (Cross-Linking and Immuno-Precipitation) in Cells with or Without the MicroRNA of Interest Depleted.

MicroRNAs (miRNAs) are short noncoding RNAs and important players in the regulation of gene expression through post-transcriptional mechanisms. MicroRNAs regulate many cellular processes and are involved in disease progression. Identification of novel miRNA-to-target RNA connections can fill the gaps in the signaling pathways and suggest new therapeutic targets. MiRNA targets are often predicted by base-complementarity of their seed and flanking sequences with target sequences. Direct targets can also be identified by the physical interaction between the miRNA and the target RNA using immunoprecipitation of the Argonaute (AGO) protein, a component of the RNA-induced silencing complex, followed by ligation of AGO-associated miRNA and target RNA and next generation sequencing (CLASH). Databases describing these miRNA-RNA interactions have been generated from cells commonly studied or used. However, because the regulation by miRNAs varies among organs, tissues, cell types and species, identifying relevant targets in specific cells under conditions of interest may not be available. Here, the author describes simplified methods of AGO2-CLASH and AGO2-CLIP to identify miRNA targets by comparing primary cells derived from wild-type mice and those from specific miRNA knockout mice.