title: Genome-Wide siRNA Screening Using Forward Transfection: Identification of Modulators of Membrane Trafficking in Mammalian Cells.
abstract: RNA interference (RNAi) has become an essential tool for molecular and cellular biologists to dissect cell function. In recent years its application has been extended to genome-wide studies, enabling the systematic identification of new cell regulation mechanisms and drug targets. In this chapter, a protocol for a genome-wide RNAi screen coupled to high-content microscopy is presented. Specifically we describe key features of assay design, plate layout, and a protocol for forward transfection of small interfering RNAs (siRNAs) in a 384-well plate format. As an example of its application in identifying modulators of membrane trafficking, we also provide a protocol to measure the efficacy of intracellular delivery of the B subunit of Shiga-like toxin to the Golgi complex. Finally we show an automated image analysis routine that can be used to extract single cell data from the screen, thereby providing a quantitative ranking of how a large panel of siRNAs affects this biological process.
authors: School of Biology and Environmental Science & UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland.
authors: Mariana G
authors: MG
authors: Bexiga
authors: School of Biology and Environmental Science & UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland. jeremy.simpson@ucd.ie.
authors: Jeremy C
authors: JC
authors: Simpson
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D016678:Genome; D007091:Image Processing, Computer-Assisted; D008322:Mammals; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D022641:Shiga Toxin 2 / Q000378:metabolism; D014162:Transfection / Q000379:methods*
chemical_list: D034741:RNA, Small Interfering; D022641:Shiga Toxin 2
keywords: Genome-wide RNAi screen; High-content microscopy; Image analysis; Mammalian cells; Membrane trafficking