In vitro RNA Cleavage Assay for Argonaute-Family Proteins

Recent studies have revealed that Argonaute proteins are crucial components of the RNA-induced silencing complexes (RISCs) that direct both small interfering RNA (siRNA)- and microRNA (miRNA)-mediated gene silencing. Full complementarity between the small RNA and its target messenger RNA (mRNA) results in RISC-mediated cleavage (“Slicing”) of the target mRNA. A subset of Argonaute proteins directly contributes to the target cleavage (“Slicer”) activity of the RISC. We describe (in vitro ) Slicer assays using endogenous Argonaute protein immunopurified from animal cells and recombinant Argonaute protein produced in and purified from Escherichia coli .