Identifying microRNA targets using AGO-RIPseq

microRNAs (miRNA) are small, noncoding RNAs that bind to messenger RNAs (mRNAs) and regulate their activity. They are, therefore, important posttranscriptional regulators. In recent years it has become clear that miRNAs regulate large genetic networks, rather than single genes, and that one gene can be targeted by several miRNAs. To understand the role of miRNAs in cells or tissues, it is therefore important to analyze the targetome of miRNAs. Here, Lund University researchers present a technique called Argonaute-RNA Immunoprecipitation (AGO-RIP) which takes advantages of the fact that miRNAs and their targets are directly bound by the Argonaute protein family. With this approach quantitative, genome-wide analysis of miRNA targets is possible.

Schematics of the AGO-RIP procedure

Cultured cells or freshly harvested tissue can be used as starting material for the AGO-RIP. Upon cell/tissue lysis, the AGO protein is immunoprecipitated using magnetic beads labeled with AGO antibodies and RNA is extracted. The quality of the RIP is controlled by determining the enrichment of miRNAs, and by the measurement of the concentration and size distribution of the RNA samples. After the quality control, cDNA libraries are prepared and small or total RNA sequencing followed by bioinformatic analyses are conducted