title: Quantitative and automated high-throughput genome-wide RNAi screens in C. elegans.
abstract: RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene (1). While the creation of libraries of RNAi clones covering most of the C. elegans genome (2,3) opened the way for true functional genomic studies (see for example (4-7)), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens (8). The approach relies on microscopic imaging and image analysis.      Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.     We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29  and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing(9). When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.
authors.affiliation: Centre d&#x2019;Immunologie de Marseille-Luminy, Universit&#233; de la M&#233;diterran&#233;e.
authors.forename: Barbara
authors.initials: B
authors.lastname: Squiban
authors.forename: Jérôme
authors.initials: J
authors.lastname: Belougne
authors.forename: Jonathan
authors.initials: J
authors.lastname: Ewbank
authors.forename: Olivier
authors.initials: O
authors.lastname: Zugasti
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000276:immunology; D055786:Gene Knockout Techniques / Q000379:methods*; D055106:Genome-Wide Association Study; D057166:High-Throughput Screening Assays / Q000379:methods*; D007113:Immunity, Innate / Q000235:genetics; D034622:RNA Interference