( gene silencing and siRNA concentration...)Delivering siRNAs to Difficult Cell Types

gene silencing and siRNA concentration, hMSC cells were electroporated with five different concentrations of GAPDH siRNA or scrambled negative control siRNA. Real-time RT-PCR revealed a dose dependent reduction in GAPDH mRNA levels (Figure 2). In these experiments and others 0.5-2.5 g siRNA (in a 75 l electroporation volume) was found to be most effective.

Varying the number of pulses is overall the most influential parameter for achieving maximum reduction of target mRNA levels. For most mammalian cell types one pulse is usually sufficient to achieve at least 70% reduction in target gene expression. In general more pulses resulted in better siRNA uptake but increased cell mortality, thus the two must be balanced. Figure 4 shows the effect that varying pulses can have when a GAPDH siRNA or a control scrambled siRNA was electroporated into NHDF-Neo primary cells.

Figure 4. GAPDH Silencing and Cell Viability vs. Number of Electroporation Pulses in Primary Cells. siRNA targeting GAPDH or a scrambled sequence (1.5 g) was electroporated into NHDF-Neo primary cells using a varying number of electroporation pulses. 24 hours post-transfection, the cells were harvested and analyzed by real-time RT-PCR for gene expression levels. 18S rRNA levels were used to normalize GAPDH mRNA levels. Percent remaining gene expression was calculated as a percentage of gene expression compared with the negative control siRNA.

Summary
Electroporation with Ambion's siPORT siRNA Electroporation Buffer and optimized conditions provides a highl
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