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Efficient Delivery of siRNAs to Human ,,, Primary Cells: Electroporation ,,, vs. Chemical Transfection

keratinocytes (NHEK) using a panel of chemical transfection reagents. Cells were harvested 48 hours post transfection and analyzed for GAPDH expression using real-time RT-PCR.

In HUVEC cells, use of siPORT Amine transfection reagent (Ambion) elicited a decrease in GAPDH gene expression by more than 85%. Reduction of GAPDH gene expression by >50% was accomplished in HUVEC cells using several siRNA transfection agents from other manufacturers (Figure 1, Panel A). In contrast, none of the transfection agents were capable of delivering enough GAPDH siRNA to NHEK cells to reduce GAPDH expression by more than 35% (Figure 1, Panel B). This example was chosen to emphasize that primary cells can vary widely in their ability to be efficiently chemically transfected, even when a wide variety of transfection agents and protocols are used.

Figure 1. Reduction of GAPDH Gene Expression in HUVEC and NHEK Cells Using Various Transfection Agents. A chemically synthesized siRNA targeting GAPDH and a scrambled siRNA sequence were individually transfected into HUVEC cells (Panel A) or NHEK cells (Panel B) using various commercially available transfection agents. Manufacturer standard protocols were used for all transfections. 48 hours post-transfection, the cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. 18S rRNA levels were used to normalize the GAPDH expression data. Percent
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