Summary

• The high-throughput, genome-scale application of RNA interference to cell-based screens — mainly in Drosophila melanogaster and mammalian cells — is revolutionizing the field of functional genomics, providing a powerful method for perturbing gene activities in cultured cells.
• As well as direct loss-of-function screening, modifier screens can be readily conducted using RNAi, making this technique particularly useful for the analysis of signal transduction pathways.
• RNAi has also become a method of choice for key steps in the development of therapeutic agents, from target discovery and validation to the analysis of the mechanisms of action of drug candidates, particularly through the use of modifier screens.
• Successful implementation of a high-throughput, cell-based RNAi screen requires sophisticated infrastructure and know-how in assay design. Choosing the appropriate cell lines, screening reagents and read-out options are crucial to a successful screen.
• In Drosophila melanogaster, all silencing reagents are based on long dsRNAs. In mammals, the choice of silencing reagent is between synthetic siRNA-like molecules, which produce only transient silencing, and vector-based shRNAs that can yield more sustained silencing.
• Two screening paradigms can be used for large-scale RNAi screens. Systematic screens target individual genes and are the most broadly applicable in terms of the phenotypes that can be studied, but raise issues of cost. Selection-based screens use pooled libraries of shRNAs and are faster, logistically simpler and less expensive; however, a selectable phenotype is required for this type of screen.
• Several important issues surround optimization and quality control in RNAi screens, including controlling RNAi specificity to avoid false positives, optimizing silencing to minimize false negatives, and ensuring the maximum robustness and sensitivity of the screen.