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High Throughput siRNA Delivery In Vitro: From Cell Lines to Primary Cells

electroporation chambers which often require a minimum of 1 x 105 cells to achieve acceptable delivery and enough viable cells for analysis, the combination of the siPORTer-96 and the siPORT siRNA Electroporation Buffer facilitated experiments with as few as 2.5 x 104 cells.

Figure 5. Reduction of GAPDH Gene Expression in Normal Human Dermal Fibroblasts-Neonatal after Electroporation using siPORTer-96 Electroporation Chamber. siRNA targeting GAPDH or a non-targeting siRNA (1.0 g) were electroporated into Normal Human Dermal FibroblastsNeonatal (an adherent primary cell type) using the siPORTer-96 Electroporation Chamber (Ambion) powered by a Bio-Rad Gene Pulser Xcell pulse generator. Samples 148 (shown) were transfected with Silencer GAPDH siRNA (Ambion) while samples 4996 were transfected with Silencer Negative Control #1 (Ambion) (data not shown). 48 h post-transfection, cells were harvested and target mRNA levels were analyzed by real-time RT-PCR. 18S rRNA levels were used to normalize GAPDH expression. Remaining Gene Expression (%) was calculated as a percentage of gene expression compared with the averaged value of cells transfected with the negative control siRNA.


Summary

Many siRNA applications require delivery of hundreds to thousands of siRNAs to cells with high efficiency while minimizing toxicity. Chemical reverse transfection with siPORT NeoFX Transfection Agent efficiently and reproducibly delivered siRNAs to immortalized, adherent cells in 96 and 384 well plates. In
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