title: High-throughput RNAi screening by time-lapse imaging of live human cells.
abstract: RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.
authors.affiliation: MitoCheck Project Group, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
authors.forename: Beate
authors.initials: B
authors.lastname: Neumann
authors.forename: Michael
authors.initials: M
authors.lastname: Held
authors.forename: Urban
authors.initials: U
authors.lastname: Liebel
authors.forename: Holger
authors.initials: H
authors.lastname: Erfle
authors.forename: Phill
authors.initials: P
authors.lastname: Rogers
authors.forename: Rainer
authors.initials: R
authors.lastname: Pepperkok
authors.forename: Jan
authors.initials: J
authors.lastname: Ellenberg
mesh_terms: D001331:Automation; D015374:Biosensing Techniques / Q000379:methods*; D002877:Chromosomes, Human / Q000235:genetics / Q000648:ultrastructure; D005453:Fluorescence; D016678:Genome; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells / Q000473:pathology / Q000648:ultrastructure; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D046228:Microarray Analysis; D008856:Microscopy, Fluorescence / Q000379:methods*; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013997:Time Factors; D014162:Transfection
chemical_list: D006657:Histones; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins