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

Solid-phase reverse trasfection on cell microarray 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 microarrays. We have developed a quick and reliable protocol for automated solid-phase reverse trasfection of human cells with siRNAs in multiwell plates complementing solid-phase reverse transfection on cell microarrays. The methos 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 ahow 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.