Optimized protocols for RNA-induced silencing complex assembly and cleavage assays

siRNAs and miRNAs guide the RNA-induced silencing complex (RISC) to regulate the expression of their target mRNA. In vitro RISC assembly and cleavage assays are proven helpful tools to characterize the biochemical properties of the RISC. Here, we provide an optimized protocol for RISC assembly and cleavage in vitro without the use of radiolabeled RNA. This protocol can directly detect RISC assembly and cleavage products within 5 days. Moreover, the methods presented here can simultaneously detect 5′- and 3′-cleavage products.