In vitro analysis of RNA interference in Drosophila melanogaster Benjamin Haley, Guiliang Tang, and Phillip D. Zamore * Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Lazare Research Building, Room 825, 364 Plantation Street, Worcester, MA 01605, USA Accepted 7 February 2003 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. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: RNA interference; RNAi; Double-stranded RNA; Small interfering RNA; siRNA; RNA degradation; Posttranscriptional gene silencing; PTGS 1. Introduction In animals, double-stranded RNA (dsRNA) induces potent and specific gene silencing by a phenomenon known as RNA interference (RNAi). When the complete sequence of an animalÕs genome is known, RNAi can be used to induce a loss-of-function phenotype for any gene, without recourse to labor-intensive, traditional genetic methods. RNAi can also be used to verify or complement existing genetic mutations. The rapid adoption of RNAi as a surrogate genetics tool stems from both its potency and its specificity [1–5]. Despite apparent differences in mechanism, the RNAi pathway is widely conserved, and RNAi has been demonstrated in such widely diverged eukaryotes as Neurospora, trypanosomes, and mammals [1,3,6–15]. However, while many researchers routinely use RNAi as a tool, the actual mechanism by which it works remains incompletely understood. RNAi is triggered by the introduction of dsRNA, typically longer than 200 bp [4]. The dsRNA is converted into 21- to 23-nt RNA duplexes, termed small interfering RNAs (siRNAs), by ATP-dependent endonucleolytic cleavage [16,17]. siRNAs are the specificity determinants of the RNAi pathway [17–19]. The RNase III enzyme, Dicer, mediates this initial cleavage event [20], yielding double-stranded siRNAs with 2-nucleotide 3 0 overhangs, 5 0 -terminal phosphates, and 3 0 hydroxyls [17,21]. The initial processing of dsRNA can be bypassed by synthetic oligonucleotides bearing the same structure as Dicer- produced siRNAs [3,17,18]. In Drosophila these siRNAs are ultimately incorporated into in an 550-kDa ribo- nucleoprotein complex (RNP) that mediates target cleavage [22,23], although a smaller complex with target- cleaving activity has also been identified [21]. This smaller complex likely corresponds to a minimal active RNA- induced silencing complex and contains single-stranded siRNA [21]. In addition, in vitro experiments have iden- tified an 360-kDa RNP that contains double-stranded siRNA but is not yet competent for mediating target cleavage [21]. Although the 360-kDa complex is inactive, its formation seems to be a prerequisite for downstream events in the RNAi pathway. 2. Preparation of translationally active Drosophila embryo lysates that recapitulate RNAi in vitro A cell-free system for studying RNAi in vitro was first described by Tuschl and co-workers in 1999 [24]. This Methods 30 (2003) 330–336 www.elsevier.com/locate/ymeth * Corresponding author. E-mail address: phillip.zamore@umassmed.edu (P.D. Zamore). 1046-2023/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1046-2023(03)00052-5