title: Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure.
abstract: RNAi using siRNA is a very powerful tool for functional genomics to identify new drug targets and biological pathways. Although their use in epithelial cells is relatively easy and straightforward, transfection in other cell types is still challenging. The authors report the optimization of transfection conditions for Raw 267.4 macrophage cells. The herein described procedure makes use of automated confocal microscopy, enhanced green fluorescent protein (EGFP)-expressing macrophages, and fluorescently labeled siRNAs to simultaneously quantify both siRNA uptake and silencing efficiency. A comparison of 10 commercial transfectants was performed, leading to the selection of the transfectant giving the highest reproducible knock-down effect without inducing cell toxicity or cell activation. Several buffers used for siRNA/lipid complex assembly were tested, and such a study revealed the crucial importance of this parameter. In addition, a kinetics study led to the determination of the optimal siRNA concentration and the best time window for the assay. In an original approach aimed at simultaneously optimizing both the high-throughput screening process and biological factors, optimal reagent volumes and a process flowchart were defined to ensure robust silencing efficiencies during screening. Such an account should pave the way for future genome-wide RNAi research in macrophages and present an optimization procedure for other "hard-totransfect" cell lines.
authors.affiliation: Biology of Intracellular Pathogens Equipe Avenir Inserm, Seoul, South Korea. jcarralot@ip-korea.org
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Carralot
authors.forename: Tae-Kyu
authors.initials: TK
authors.lastname: Kim
authors.forename: Boris
authors.initials: B
authors.lastname: Lenseigne
authors.forename: Annette S
authors.initials: AS
authors.lastname: Boese
authors.forename: Peter
authors.initials: P
authors.lastname: Sommer
authors.forename: Auguste
authors.initials: A
authors.lastname: Genovesio
authors.forename: Priscille
authors.initials: P
authors.lastname: Brodin
mesh_terms: D000818:Animals; D001331:Automation / Q000295:instrumentation / Q000379:methods / Q000592:standards; D002138:Calibration; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004355:Drug Stability; D055785:Gene Knockdown Techniques / Q000379:methods* / Q000592:standards; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D009994:Osmolar Concentration; D034741:RNA, Small Interfering / Q000032:analysis / Q000302:isolation & purification* / Q000494:pharmacology; D014162:Transfection / Q000295:instrumentation / Q000379:methods* / Q000592:standards
chemical_list: D034741:RNA, Small Interfering