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siRNA Design: It's All in the Algorithm

ured by qRT-PCR and normalized against 18S rRNA from samples harvested 48 hr after siRNA transfection into HeLa cells.


In subsequent analyses of sets of multiple siRNAs targeting the same transcripts, Cenix noted that certain genes, and in some cases certain transcripts, appear significantly more refractory to siRNA-based silencing than the majority (data not shown). Since it was not known what percent of genes would fall into this category, it became clear that any extrapolations of silencing success rates from tens or even hundreds of siRNAs would be of quite limited statistical value. Thus, expecting that the eventual "true" success rate of its algorithm would likely be lower than the 94% initially observed with the 79 kinases, Cenix extended the same performance analysis to a much larger set of siRNAs to create a more statistically relevant data set. Figure 2 shows the distribution of the siRNA efficacy for over 1,100 siRNAs targeting nearly 400 endogenously-expressed human transcripts.

The comprehensive survey indicated that when three Cenix designed siRNAs per gene were tested, one or more of the siRNAs achieved >70% silencing for over 93% of tested genes, >80% silencing for nearly 80% of tested genes, and >90% silencing for approximately half of tested genes. On a per siRNA basis, approximately 80% of the individual siRNAs showed >70% silencing of their target. The performance data clearly confirm the success of the Ceni
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