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siRNA Design: It's All in the Algorithm

using 18S rRNA as the loading control. The reduction in target mRNA is calculated by comparing the PCR results for each test siRNA versus a negative control siRNA. Even extremely low concentrations of Cenix pre-designed siRNAs elicit a strong RNAi response.


There are several advantages to using low concentrations of highly active siRNAs; the most important is minimization of off-target effects, as these are known to increase with increasing concentrations of input siRNA [9, 10]. Off-target effects from high concentrations of siRNAs in the cells are likely to result because more of the sense strand becomes bound to the RISC and degrades transcripts with sequence complementary to the sense strand siRNA, or dsRNA binding proteins that stimulate antiviral response pathways are more likely to be activated and induce the expression of antiviral response genes. Another important advantage to using less siRNA for silencing experiments is that transfecting lower concentrations of siRNAs allows multiple siRNAs to be transfected at the same time. Ambion researchers have simultaneously reduced the expression of as many as five genes using highly active siRNAs designed with the Cenix algorithm (Figure 4).

Figure 4. Multigene Knockdown with siRNAs. Equal amounts of five different siRNAs were mixed and the indicated concentrations of siRNA were transfected into HeLa cells. 48 hours post-transfection, RNA from the cells was recovered and analyzed for target mRNA reduction by real-time PCR, using 18S rRNA as the loading control. The reduction in target mRNA is calculated by comparing the PCR results for each test siRNA versus a negat
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