Reverse transfection on cell arrays for high content screening microscopy

Holger Erfle; Beate Neumann; Urban Liebel; Phill Rogers; Michael Held; Thomas Walter; Jan Ellenberg; Rainer Pepperkok

doi:10.1038/nprot.2006.483

Reverse transfection on cell arrays for high content screening microscopy

Nat Protoc. 2007;2(2):392-9. doi: 10.1038/nprot.2006.483.

Authors

Holger Erfle¹, Beate Neumann, Urban Liebel, Phill Rogers, Michael Held, Thomas Walter, Jan Ellenberg, Rainer Pepperkok

Affiliation

¹ MitoCheck Project Group, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. erfle@embl.de

PMID: 17406600
DOI: 10.1038/nprot.2006.483

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cells, Cultured
Fluorescent Antibody Technique / methods*
Humans
RNA Interference*
RNA, Small Interfering / genetics*
Tissue Array Analysis / methods*
Transfection / methods*

Substances

RNA, Small Interfering