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Journal of Biomolecular Screening
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Work Flow for Multiplexing siRNA Assays by Solid-Phase Reverse Transfection in Multiwell Plates

Holger Erfle

Cell Biology/Biophysics Unit, EMBL, Heidelberg, Germany, holger.erfle{at}bioquant.uni-heidelberg.de

Beate Neumann

MitoCheck Project Group, EMBL, Heidelberg, Germany

Phill Rogers

MitoCheck Project Group, EMBL, Heidelberg, Germany

Jutta Bulkescher

MitoCheck Project Group, EMBL, Heidelberg, Germany

Jan Ellenberg

Gene Expression Unit, EMBL, Heidelberg, Germany

Rainer Pepperkok

Cell Biology/Biophysics Unit, EMBL, Heidelberg, Germany

Solid-phase reverse transfection on cell microarrays is a high-throughput method for the parallel transfection of mammalian cells. However, the cells transfected in this way have been restricted so far to microscopy-based analyses. Analysis methods such as reverse transcriptase—polymerase chain reaction (RT-PCR) and access to higher cell numbers for statistical reasons in microscopy-based assays are not possible with solid-phase reverse transfection on cell microarrays. We have developed a quick and reliable protocol for automated solid-phase reverse transfection of human cells with siRNAs in multiwell plates complementing solid-phase reverse transfection on cell microarrays. The method retains all advantages of solid-phase reverse transfection such as long-term storage capacity after fabrication, reduced cytotoxicity, and reduced cost per screen compared with liquid-phase transfection in multiwell plates. The protocol has been tested for the RNAi-mediated knockdown of several genes in different cell lines including U20S, RPE1, A549, and HeLa cells. We show that even 3 months after production of the "ready to transfect" multiwell plates, there is no reduction in their transfection efficiency as assessed by RT-PCR and nuclear phenotyping by fluorescence microscopy. We conclude that solid-phase reverse transfection in multiwell plates is a cost-efficient and flexible tool for multiplexing cellular assays. (Journal of Biomolecular Screening. 2008:575-580)

Key Words: solid-phase reverse transfection • siRNA

This version was published on August 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 7, 575-580 (2008)
DOI: 10.1177/1087057108320133
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