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

expression was calculated as a percentage of gene expression compared to the negative control siRNA. Duplicates were performed for each sample.


Electroporation
An alternative to chemical transfection-mediated nucleic acid delivery is electroporation. This method involves applying an electric field pulse to induce the formation of microscopic pores (electropores) in the cell membrane. This allows molecules, ions, and water to traverse the membrane. Under specific pulse conditions, the electropores reseal and the "electroporated" cells recover and continue to grow. A distinct advantage of electroporation is that it is not dependent on cell division, and reduction in gene expression can be detected a few hours after nucleic acid delivery. This is important when working with primary cells since most divide relatively slowly if at all.

We used electroporation (Nucleofector, Amaxa) to deliver Cy3-labeled siRNA targeting GAPDH into HUVEC cells. A HUVEC-specific protocol (Amaxa) was used. We were pleasantly surprised to find that almost every cell had taken up detectable amounts of Cy3-labeled siRNA (Figure 2). The Nucleofector was then used to electroporate HUVEC, NHEK, and normal human dermal fibroblasts neonatal (NHDF-Neo) with five different concentrations of GAPDH siRNA. Real time RT-PCR revealed a dose dependent reduction in GAPDH expression in all three cell types. Some toxicity was observed at the highest siRNA dose tested (2.66 g), but there was a complete absence of non-specific effects at 1.33 g siRNA (Figure 3). To ex
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PKC Sampler Kit 10 mu g each...
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