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In vitro analysis of RNA interference in Drosophila melanogaster
Benjamin Haley1, Guiliang Tang, Phillip D Zamore
1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Lazare Research Building, Room 825, 364 Plantation Street, Worcester, MA 01605, USA.
Abstract:
Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.