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Reverse transfection on cell arrays for high content screening microscopy.

Nature protocols (2007-04-05)
Holger Erfle, Beate Neumann, Urban Liebel, Phill Rogers, Michael Held, Thomas Walter, Jan Ellenberg, Rainer Pepperkok
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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Silica gel orange, with moisture indicator free of heavy metals
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, microbiologically tested, ≥96% (agarose gel electrophoresis)

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