Detection of MicroRNAs Released from Argonautes.

ABSTRACT

The Argonaute (AGO) family of proteins plays an essential role in the process of microRNA (miRNA)-mediated gene silencing. More specifically, they are the only known proteins to associate directly with miRNAs within the RNA-induced silencing complex (RISC). Given the importance of miRNA regulation of the transcriptome and its vast implications for human disease, it is essential to understand the molecular underpinnings of miRNA-AGO interactions. Although there are methods available to investigate mature miRNA decay and loading onto AGO2, no feasible method exists to detail the opposite process release of miRNA from associated AGO proteins. In this chapter, we describe in detail a methodology derived from biochemical approaches, which can be used to quantify the release of any given miRNA from AGOs.