Maximizing Silencing Efficacy
In addition to increasing the likelihood that an siRNA will be active, the Cenix algorithm results in siRNAs that are remarkably potent (Figure 2). Using Cenix-designed siRNAs in optimized transfection systems, Ambion researchers routinely observe greater than 90% reduction in target mRNA levels with nM or even pM amounts of siRNA. Figure 3 shows an experiment in which siRNAs targeting three different genes were transfected at different concentrations. With one exception, each of the siRNAs performed essentially the same when transfected at either 200 pM or 10 nM. The observation that GAPDH siRNA was less effective at 200 pM than at 1 nM is probably because GAPDH mRNA is significantly more abundant in cells than the other two mRNA targets tested. It is probable that the concentration of GAPDH transcripts in the cell exceed the concentration of RISC-associated GAPDH siRNA generated by transfecting only 200 pM GAPDH siRNA.
Figure 2 . Distribution of Gene Silencing Measured for 1,106 siRNAs Targeting 379 Endogenously-expressed Human Genes. Target mRNA levels were measured by qRT-PCR, normalized against 18S rRNA from samples harvested 48hr after siRNA transfection into HeLa cells. Percent of genes exhibiting siRNA-induced silencing above the noted thresholds (F70=70%, F80=80%, etc.) are shown.
Efficacy of Cenix-designed siRNAs. siRNAs targeting three different genes
were transfected into HeLa cells. 48 hours post-transfection, RNAs from
the cells were recovered and analyzed for target mRNA by real-time PCR,