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Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. The development of siRNA libraries targeting entire classes of human genes now provides an opportunity to carry out genome scale RNAi screens to discover and characterize gene functions directly in human cells. However, methods for high throughput delivery, necessary for RNAi screening at the genome scale, have not been sufficiently developed and are limited by cell lines. Here we describe high throughput siRNA delivery methods to facilitate experiments with both immortalized and primary cells. We have adapted chemical reverse transfection for immortalized adherent cell lines in 96 well format. Reverse transfection allows for rapid siRNA library screening in cultured mammalian cell lines and may provide improved reliability for transfections performed in 96 or 384 well plates and improved efficiency for transfections performed in high throughput manner. For cell types that are recalcitrant to lipofection-based methods, we have found transfection via electropermeabilization (electroporation) to be a valuable alternative. We have developed a 96 well electroporation device to facilitate high throughput siRNA delivery to primary cells. Successful gene silencing and high cell viability was achieved in 7 cell types: normal human dermal fibroblasts - neonatal (NHDF-Neo), renal proximal tubule cells (RPTEC), human mesenchymal stem cells (hMSC), normal human umbilical vein endothelial cells (HUVEC), mouse embryonic fibroblasts (MEF), rhesus monkey stem cells (RMSC), and acute T-cell leukemia (Jurkat) cells. In all cases, target gene expression is reduced within the population by greater than 70%. A 96 well electroporation device allows screening of large numbers of siRNAs rapidly, reproducibly, and in parallel. Large scale RNAi screening in most biologically-relevant primary cells, a setting where genomic manipulations have proven difficult, is a valuable research tool for studying gene function analysis, target validation, and gene therapeutic approaches.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]