title: An efficient and fully automated high-throughput transfection method for genome-scale siRNA screens.
abstract: RNA interference (RNAi), combined with the availability of genome sequences, provides an unprecedented opportunity for the massive and parallel investigations of gene function. Small interfering RNA (siRNA) represents a popular and quick approach of RNAi for in vitro loss-of-function genetic screens. Efficient transfection of siRNA is critical for unambiguous interpretation of screen results and thus overall success of any siRNA screen. A high-throughput, lipid-based transfection method for siRNA was developed that can process eighty 384-well microplates in triplicate (for a total of 30,720 unique transfections) in 8 h. Transfection throughput was limited only by the speed of robotics, whereas the cost of screening was reduced. As a proof of principle, a genome-scale screen with a library of 22,108 siRNAs was performed to identify the genes sensitizing cells to mitomycin C at concentrations of 0, 20, and 60 nM. Transfection efficiency, performances of control siRNAs, and other quality metrics were monitored and demonstrated that the new, optimized transfection protocol produced high-quality results throughout the screen.
authors.affiliation: Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA 19454, USA. namjin.chung@bms.com
authors.forename: Namjin
authors.initials: N
authors.lastname: Chung
authors.forename: Louis
authors.initials: L
authors.lastname: Locco
authors.forename: Kevin W
authors.initials: KW
authors.lastname: Huff
authors.forename: Steven
authors.initials: S
authors.lastname: Bartz
authors.forename: Peter S
authors.initials: PS
authors.lastname: Linsley
authors.forename: Marc
authors.initials: M
authors.lastname: Ferrer
authors.forename: Berta
authors.initials: B
authors.lastname: Strulovici
mesh_terms: D000465:Algorithms; D001331:Automation; D004526:Efficiency; D020869:Gene Expression Profiling / Q000379:methods; D015894:Genome, Human / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D034741:RNA, Small Interfering / Q000494:pharmacology*; D014162:Transfection / Q000295:instrumentation / Q000379:methods*
chemical_list: D034741:RNA, Small Interfering