In siRNA delivery the siRNA sequence may impose non-specific and off-target effects in the cells. Lowering the siRNA concentration significantly reduces the risk of unwanted off-target effects. With NIMT®FeOfection|PURPLE a high gene silencing effect is maintained when transfecting with siRNA concentrations as low as 5 nM. This was determined both at the mRNA level (Figure 1) and at the protein level (Figure 2).
Experimental set-up:
786-O (human renal cell adenocarcinoma) cells were transfected with Gapdh siRNA or a negative control siRNA. The mRNA levels were assessed with real-time PCR 24 hours post transfection using ß-actin as endogenous control. The GAPDH enzyme activity was determined 48 hours post transfection with KDalert Enzyme assay (Ambion).
786-O cells were reverse transfected with 7000 cells/well in a 96-well plate. 4.2 µl NIMT®FeOfection|PURPLE and 0.5-80 nM siRNA were used/well. The percent knockdown was calculated as the relative quantity of GAPDH in Gapdh siRNA treated cells as compared to that of cells treated with negative control siRNA.
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