Work flow for multiplexing siRNA assays by solid-phase reverse transfection in multiwell plates.
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.
Erfle
Holger
H
Cell Biology/Biophysics Unit, EMBL, Heidelberg, Germany. holger.erfle@bioquant.uni-heidelberg.de
Neumann
Beate
B
Rogers
Phill
P
Bulkescher
Jutta
J
Ellenberg
Jan
J
Pepperkok
Rainer
R
D001331:Automation; D001681:Biological Assay / Q000379:methods; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D008853:Microscopy; D008856:Microscopy, Fluorescence; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods*
D034741:RNA, Small Interfering