title: Reverse transfection on cell arrays for high content screening microscopy.
abstract: Here, we describe a robust protocol for the reverse transfection of cells on small interfering (siRNA) arrays, which, in combination with multi-channel immunofluorescence or time-lapse microscopy, is suitable for genome-wide RNA interference (RNAi) screens in intact human cells. The automatic production of 48 'transfection ready' siRNA arrays, each containing 384 samples, takes in total 7 h. Pre-fabricated siRNA arrays can be used without loss of transfection efficiency at least up to 15 months after printing. Different human cell lines that have been successfully transfected using the protocol are presented here. The present protocol has been applied to two genome-wide siRNA screens addressing mitosis and constitutive protein secretion.
authors.affiliation: MitoCheck Project Group, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. erfle@embl.de
authors.forename: Holger
authors.initials: H
authors.lastname: Erfle
authors.forename: Beate
authors.initials: B
authors.lastname: Neumann
authors.forename: Urban
authors.initials: U
authors.lastname: Liebel
authors.forename: Phill
authors.initials: P
authors.lastname: Rogers
authors.forename: Michael
authors.initials: M
authors.lastname: Held
authors.forename: Thomas
authors.initials: T
authors.lastname: Walter
authors.forename: Jan
authors.initials: J
authors.lastname: Ellenberg
authors.forename: Rainer
authors.initials: R
authors.lastname: Pepperkok
mesh_terms: D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D046888:Tissue Array Analysis / Q000379:methods*; D014162:Transfection / Q000379:methods*
chemical_list: D034741:RNA, Small Interfering