Work flow for multiplexing siRNA assays by solid-phase reverse transfection in multiwell plates.

Cell Biology/Biophysics Unit, EMBL, Heidelberg, Germany.
Journal of Biomolecular Screening (Impact Factor: 2.01). 08/2008; 13(7):575-80. DOI: 10.1177/1087057108320133
Source: PubMed

ABSTRACT 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.

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Available from: Beate Neumann, Apr 13, 2015
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