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