Ji Won Lee, Chang Hoon Shin, J Grayson Evans, Erick C Won, Je-Hyun Yoon, Kyung-Won Min
The Argonaute (AGO) family of proteins is fundamental to microRNA (miRNA)-mediated gene silencing, serving as the central component of the RNA-induced silencing complex (RISC) by forming direct and stable binding with mature miRNAs. While miRNA-AGO interactions are essential for controlling gene expression and closely linked to human disease, AGO-free mature miRNAs also contribute to fine-tuning gene expression regulation through direct interaction with RNA-binding proteins (RBPs). This chapter presents a detailed biochemical methodology to quantify single-stranded mature miRNAs dissociated from AGO2, providing an approach to studying miRNAs release mechanisms.