Advanced ReviewRNAi screening: new approaches,understandings, and organismsStephanie E. Mohr1and Norbert Perrimon2∗RNA interference (RNAi) leads to sequence-specific knockdown of gene function.The approach can be used in large-scale screens to interrogate function in variousmodel organisms and an increasing number of other species. Genome-scale RNAiscreens are routinely performed in cultured or primary cells or in vivo in organismssuch as C. elegans. High-throughput RNAi screening is benefitting from thedevelopment of sophisticated new instrumentation and software tools for collectingand analyzing data, including high-content image data. The results of large-scaleRNAi screens have already proved useful, leading to new understandings ofgene function relevant to topics such as infection, cancer, obesity, and aging.Nevertheless, important caveats apply and should be taken into considerationwhen developing or interpreting RNAi screens. Some level of false discoveryis inherent to high-throughput approaches and specific to RNAi screens, falsediscovery due to off-target effects (OTEs) of RNAi reagents remains a problem.The need to improve our ability to use RNAi to elucidate gene function at largescale and in additional systems continues to be addressed through improved RNAilibrary design, development of innovative computational and analysis tools andother approaches. 2011 John Wiley & Sons, Ltd. WIREs RNA 2011 DOI: 10.1002/wrna.110INTRODUCTIONRNA interference (RNAi) is a conserved endoge-nous activity1that can be harnessed as a toolfor functional genomics studies.2–8With RNAi, gene-specific reagents are introduced into cells, trigger-ing ‘knockdown’ or reduction of gene function viasequence-specific degradation and translational inter-ference of mRNA transcripts. RNAi screening pro-vides a powerful reverse-genetic approach to large-scale functional analysis in cultured cells and in anincreasing number of in vivo systems. Like geneticscreening, RNAi screening allows for identificationof genes relevant to a given pathway, structure orfunction via association of a mutant phenotype withgene knockdown. Like chemical screening, RNAiscreening is amenable to miniaturization and automa-tion, facilitating high-throughput studies. Becauseof, at least in part, the ease of delivery of RNAi∗Correspondence to: [email protected] RNAi Screening Center, Department of Genetics,Harvard Medical School, Boston, MA, USA2Department of Genetics, Harvard Medical School and HowardHughes Medical Institutes, Boston, MA, USADOI: 10.1002/wrna.110reagents and resources available, Caenorhabditis ele-gans, Drosophila cells and mammalian cells have beenthe most-used systems for RNAi screening. Indeed,screens in these systems have already led to importantnew insights into a wide variety topics, including infec-tious disease, cancer, signaling, and aging.2,3,6,8–16Moreover, RNAi screening has benefitted from inputfrom a variety of other fields, in particular engineeringand computer science, for example, to improve meth-ods for automated high-content image acquisition andanalysis.17Over the years, researchers have gained a betterunderstanding of best practices for RNAi screen-ing, both through performing screens and throughstudy of endogenous RNAi pathways. In particular,recent improvements and refinements in methods forin vivo RNAi screening in Drosophila and mice haveopened the doors to an increasing number of large-scale in vivo studies in those systems.3RNAi has beenevolutionarily conserved and thus, it is being used tostudy an increasing number species for which func-tional genomics would otherwise not be feasible.3,18–22Despite all this progress, however, the problem of off-target effects and other sources of false discoveryremain ongoing challenges. Improvements in reagentdesign, reagent delivery, assay design and data analysis 2011 John Wiley & Sons, Ltd.Advanced Reviewwires.wiley.com/rnahave increased the quality of RNAi screen results inrecent years. However, the picture remains complexin terms of understanding and addressing all possiblesources of false positive and false negative results.23,24Despite these caveats, RNAi screening remains a pow-erful method-of-choice for genome-scale interrogationof gene function in an increasing number of sys-tems, and the results of RNAi screens continue toprovide new insights into diverse topics in biologyand biomedicine. Below, we provide an overview ofRNAi screening in cells and in vivo, focusing onnew developments and results, as well as innova-tions stemming from interaction with other fields ofstudy.RNAi SCREENING IN CELLSWhy screen in cultured cells?RNAi technology opened the doors to performingfunctional genomics in human cells and other typesof cultured and primary cells. Cell-based RNAiscreening builds upon established instrumentation,assays and other methods previously developed forchemical screening in cells. Overall, cell-based RNAiscreening provides a relatively rapid and accessibleplatform for genome-scale functional studies.2,4,5,7Alarge number of RNAi screens has been performedin Drosophila and mammalian cultured cells.2Morerecently, researchers have developed methods forscreening neuronal and muscle primary cells derivedfrom dissociated Drosophila embryos,25–27as wellas primary Drosophila haemocytes.28In addition, anincreasing number of studies are being performedusing mammalian stem cells (reviewed in Refs 29and 30). The availability of transcriptome data fortissues, tumors and cell lines, made possible by next-generation sequencing technologies, is likely to shapechoices and interpretation of cell-based RNAi screendata.5,15,23,31For example, transcriptome data mayhelp us to understand the extent to which networkspresent in a cell line reflect what is happening in vivo,and detection of single-nucleotide polymorphisms(SNPs) can reveal mismatches to reagents that arerelevant to the interpretation of results.Reagent Libraries for Cell-Based Screeningin Drosophila and Mammalian CellsRNAi screening relies on the availability of genome-wide or other large-scale RNAi reagent libraries, withone or more unique RNAi reagent directed againsteach target gene. The appropriate RNAi reagentlibrary for cell-based screening depends upon the celltype, approach and method of reagent delivery.7InDrosophila cells, the lack of an interferon responseand ability of most cell types to take up the reagent insolution makes it possible