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.小干扰RNA（siRNAs）与微RNA（miRNAs）可引导RNA诱导沉默复合体（RISC），调控其靶信使RNA（mRNA）的表达。经证实，体外RISC组装与切割实验是表征RISC生化特性的有效工具。本文提供一套无需使用放射性标记RNA的优化体外RISC组装及切割实验方案。该方案可在5天内直接检测RISC组装产物与切割产物，且可同时检测5'端与3'端切割产物。