RNA Interference (RNAi) Screening in Cultured Drosophila Cells

Genetic perturbation assays have been crucial to the discovery of molecular pathways that drive diverse biological processes. RNA interference (RNAi)-mediated depletion of gene products represents a powerful means of elucidating gene function, as it allows one to systematically probe the phenotypic effects resulting from the functional loss of specific targets. The relative ease of use of RNAi technologies in cultured cells has allowed the design and implementation of genome-wide investigations to systematically reveal gene function. In this chapter, we describe methods for high-throughput RNAi-mediated loss-of-function studies in cultured cells of Drosophila melanogaster. First, we describe the in vitro synthesis of double stranded RNAs (dsRNAs) from a genome-wide Drosophila RNAi library. Next, we outline the procedures used to carry out high-throughput RNAi screens using a cell bathing approach and high-content screening microscopy, illustrating how these experiments can be utilized to study specific cellular contexts, such as cellular stress. Finally, we illustrate some approaches commonly employed to validate the depletion of identified gene candidates.