( he optimized amount of cells (3 x 10 4 ...)Optimize Transfection of siRNAs for RNAi

he optimized amount of cells (3 x 10⁴ cells/well) and 3 l transfection reagent to establish the concentration resulting in a >50% reduction in mRNA expression. The most significant siRNA activity was attained using siRNA concentrations between 30 and 100 nM (Figure 5). We were able to achieve approximately 75%-90% reduction in GAPDH mRNA levels over control with no apparent cytotoxicity.

Figure 5. Determination of Optimal Amount of siRNA. COS-7 cells were plated at the optimized cell plating density (3.0 x10⁴ cells per well) in a 24 well dish. 24 hours after plating, cells were transfected with 100, 30, 10, or 3 nM chemically syntheesized GAPDH siRNA or negative control siRNA using the optimized volume of transfection agent (3 L per well). 48 hours following transfection, the cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. Percent gene expression was calculated as a percentage of gene expression compared to the negative control siRNA.

Note that it is common to see relative variations in data when conducting tissue culture experiments. Reproducibility can be achieved by rigorously following protocols.

siPORT Amine is manufactured for Ambion by Mirus.

TaqMan is a registered trademark of Applied Biosystems.

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REFERENCES

1. Byrom M, Pallotta V, Brown D, Ford L (2002) Visualizing siRNA in Mammalian cells: Fluorescence analysis of the RNAi ef
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