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Designing a Better siRNA


The siRNA design algorithm designed by Cenix BioScience, with whom Ambion has partnered to develop a genome wide siRNA library, represents a major improvement over the design rules first described by Tuschl and colleagues. On average, ~50% of the siRNAs featuring the Tuschl design rules (target sites beginning with AA, 3' UU overhangs for both the sense and antisense siRNA strands, ~50% G/C content; 1-3) provide at least 50% reduction in target gene expression. In contrast, in an initial screen of 79 human genes, 94% of siRNAs designed by Cenix provided greater than 70% reduction in target mRNA levels (Figure 1).

Figure 1.The Effectiveness of Cenix Designed siRNAs. siRNAs targeting 79 human genes were designed using the algorithm developed by Cenix. The top siRNA candidate for each target was prepared and transfected at a 100 nM final concentration. Forty eight hours post-transfection, target gene expression was quantified by real-time RT-PCR. Relative reduction in mRNA expression was measured against cells transfected with a negative control siRNA. Sample size was normalized by measuring 18S rRNA in the various samples using a real-time PCR.


To develop a rational siRNA design algorithm, Cenix and Ambion tested multiple siRNAs targeting hundreds of different human genes that were expressed at detectable levels in several different cell lines. More than 900 siRNAs have been designed and tested to date. The effective and ineffective siRNAs were used to judge the impact of a number of physical characteristics on siRNA potency and specificity, including Tm, nucleotide content of the 3' overhangs, siRNA length, nucleotid
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