( et gene expression at compar...)Inducing RNAi with siRNA Cocktails Generated by RNase III

et gene expression at comparable levels to a chemically synthesized siRNA targeting GAPDH (Figure 2C). This experiment demonstrates that the smaller sized siRNA cocktails produced by RNase III reduce target gene expression upon transfection into mammalian cells and suggests that altering the digestion or purification conditions to generate longer products is unnecessary for the efficient reduction of target gene expression.

Figure 2. 12-15 bp RNase III Digestion Products Elicit Silencing. A 200 bp GAPDH dsRNA (30 g) was digested with RNase III (30 U) for 1 hour at RT. Digestion products were run on a 15% non-denaturing acrylamide gel and the 12-15 bp products were excised, eluted, and ethanol precipitated. A sample was run on a 15% non-denaturing acrylamide gel for visualization (2A). HeLa cells were transfected with 100 nM of the 12-15 bp RNase III generated GAPDH siRNAs or a 21 bp chemically synthesized GAPDH siRNA. GAPDH protein levels were monitored by immunofluorescence 48 hours after transfection (2B) and the resulting images were quantitated (2C).

Specificity of Gene Silencing. We next analyzed the specificity of the siRNA for reducing target gene expression. HeLa cells were transfected with an RNase III generated siRNA population to GAPDH, and the resulting expression levels of GAPDH and a number of nonspecific target genes (La, Ku-70, c-myc, -actin, and cdk-2) were compared in transfected and nontransfected cells. Figure 3 shows a 63% reduction in GAPDH levels but no detectable reduction in the other genes examined. These data s
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