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Advanced Review RNAi screening new approaches understandings and organisms Stephanie E Mohr1 and 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 various model organisms and an increasing number of other species Genome scale RNAi screens are routinely performed in cultured or primary cells or in vivo in organisms such as C elegans High throughput RNAi screening is benefitting from the development of sophisticated new instrumentation and software tools for collecting and analyzing data including high content image data The results of large scale RNAi screens have already proved useful leading to new understandings of gene function relevant to topics such as infection cancer obesity and aging Nevertheless important caveats apply and should be taken into consideration when developing or interpreting RNAi screens Some level of false discovery is inherent to high throughput approaches and specific to RNAi screens false discovery 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 large scale and in additional systems continues to be addressed through improved RNAi library design development of innovative computational and analysis tools and other approaches 2011 John Wiley Sons Ltd WIREs RNA 2011 DOI 10 1002 wrna 110 INTRODUCTION R NA interference RNAi is a conserved endogenous activity1 that can be harnessed as a tool for functional genomics studies 2 8 With RNAi genespecific reagents are introduced into cells triggering knockdown or reduction of gene function via sequence specific degradation and translational interference of mRNA transcripts RNAi screening provides a powerful reverse genetic approach to largescale functional analysis in cultured cells and in an increasing number of in vivo systems Like genetic screening RNAi screening allows for identification of genes relevant to a given pathway structure or function via association of a mutant phenotype with gene knockdown Like chemical screening RNAi screening is amenable to miniaturization and automation facilitating high throughput studies Because of at least in part the ease of delivery of RNAi Correspondence to perrimon receptor med harvard edu 1 Drosophila RNAi Screening Center Department of Genetics Harvard Medical School Boston MA USA 2 Department of Genetics Harvard Medical School and Howard Hughes Medical Institutes Boston MA USA DOI 10 1002 wrna 110 reagents and resources available Caenorhabditis elegans Drosophila cells and mammalian cells have been the most used systems for RNAi screening Indeed screens in these systems have already led to important new insights into a wide variety topics including infectious disease cancer signaling and aging 2 3 6 8 16 Moreover RNAi screening has benefitted from input from a variety of other fields in particular engineering and computer science for example to improve methods for automated high content image acquisition and analysis 17 Over the years researchers have gained a better understanding of best practices for RNAi screening both through performing screens and through study of endogenous RNAi pathways In particular recent improvements and refinements in methods for in vivo RNAi screening in Drosophila and mice have opened the doors to an increasing number of largescale in vivo studies in those systems 3 RNAi has been evolutionarily conserved and thus it is being used to study an increasing number species for which functional genomics would otherwise not be feasible 3 18 22 Despite all this progress however the problem of offtarget effects and other sources of false discovery remain ongoing challenges Improvements in reagent design reagent delivery assay design and data analysis 2011 Jo h n Wiley So n s L td wires wiley com rna Advanced Review have increased the quality of RNAi screen results in recent years However the picture remains complex in terms of understanding and addressing all possible sources of false positive and false negative results 23 24 Despite these caveats RNAi screening remains a powerful method of choice for genome scale interrogation of gene function in an increasing number of systems and the results of RNAi screens continue to provide new insights into diverse topics in biology and biomedicine Below we provide an overview of RNAi screening in cells and in vivo focusing on new developments and results as well as innovations stemming from interaction with other fields of study RNAi SCREENING IN CELLS Why screen in cultured cells RNAi technology opened the doors to performing functional genomics in human cells and other types of cultured and primary cells Cell based RNAi screening builds upon established instrumentation assays and other methods previously developed for chemical screening in cells Overall cell based RNAi screening provides a relatively rapid and accessible platform for genome scale functional studies 2 4 5 7 A large number of RNAi screens has been performed in Drosophila and mammalian cultured cells 2 More recently researchers have developed methods for screening neuronal and muscle primary cells derived from dissociated Drosophila embryos 25 27 as well as primary Drosophila haemocytes 28 In addition an increasing number of studies are being performed using mammalian stem cells reviewed in Refs 29 and 30 The availability of transcriptome data for tissues tumors and cell lines made possible by nextgeneration sequencing technologies is likely to shape choices and interpretation of cell based RNAi screen data 5 15 23 31 For example transcriptome data may help us to understand the extent to which networks present in a cell line reflect what is happening in vivo and detection of single nucleotide polymorphisms SNPs can reveal mismatches to reagents that are relevant to the interpretation of results Reagent Libraries for Cell Based Screening in Drosophila and Mammalian Cells RNAi screening relies on the availability of genomewide or other large scale RNAi reagent libraries with one or more unique RNAi reagent directed against each target gene The appropriate RNAi reagent library for cell based screening depends upon the cell type approach and method of reagent delivery 7 In Drosophila cells the lack of an interferon response and ability of most cell types to take up the reagent in solution makes it possible to use in vitro synthesized long

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