Slicer activity in Drosophila melanogaster S2 extract

Abstract

Reconstitution of RNA-inducing silencing complex (RISC) in vitro is a powerful biochemical technique to analyze crucial steps in RNA interference (RNAi) pathways. RISC contains an RNase enzyme, Argonaute, which is guided by small interfering RNA (siRNA) to recognize and silence its targets. Drosophila S2 cell extract is a good source of enzymes and factors that faithfully recapitulate essential steps in RISC assembly and function. In this chapter, we will describe how to prepare enzymatically active cell-free S2 extract to analyze the Slicer activity of RISC as well as the effects of viral RNAi suppressors which block this process.