Reverse transfection on cell arrays for high content screening microscopy.
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.
Erfle
Holger
H
MitoCheck Project Group, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. erfle@embl.de
Neumann
Beate
B
Liebel
Urban
U
Rogers
Phill
P
Held
Michael
M
Walter
Thomas
T
Ellenberg
Jan
J
Pepperkok
Rainer
R
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*
D034741:RNA, Small Interfering