In addition, the Silencer Select siRNA design uses a chemical modification format that results in higher specificity knockdown and fewer off-target effects. Taken together, maximum knockdown is achieved with lower concentrations of siRNA.

Novel Algorithm

Applied Biosystems has developed a novel siRNA prediction algorithm based on the Support Vector Machine (SVM) artificial intelligence platform. siRNAs designed using this new algorithm and synthesized with novel chemical modifications to enhance their specificity exhibit higher overall potency compared to siRNAs predicted by previous algorithms. These highly potent siRNAs result in effective target knockdown at as much as 10-fold lower concentrations than older generation siRNAs.

Achieve Strong Knockdown with Less siRNA

The majority of Silencer Select siRNAs will induce ≥70% knockdown at concentrations ≤5 nM in cells that are easily transfected by traditional lipid-based strategies. Figure 1 shows knockdown of mRNA encoding the heavy chain for clathrin (CLTC) using three Silencer Select siRNAs designed using the new SVM-derived algorithm and synthesized with specificity-enhancing chemical modifications. These Silencer Select siRNAs demonstrate >80% CLTC knockdown at both high (100 nM) and low (3 nM) siRNA concentrations. These results indicate the exceptional potency of Silencer Select siRNAs.


Figure 1. Potency of Ambion Silencer Select siRNAs Targeting CLTC Gene. Three Silencer Select siRNAs targeting human CLTC were reverse transfected into HeLa cells in triplicate at 5 different concentrations in 24-well plates using 1.5 uL siPORT™ NeoFX™ Transfection Agent. 24 hr post-transfection, RNA was isolated from cells using the MagMAX™-96 Total RNA Isolation Kit. cDNA was synthesized with the Applied Biosystems High Capacity cDNA Reverse Transcription Kit and was used to amplify CLTC mRNA in RT-PCR reactions with CLTC Hs00191535_m1 TaqMan Gene Expression Assay primers. Percent gene expression remaining was expressed as the relative amount of CLTC mRNA in cultures transfected with CLTC targeting siRNAs versus cells transfected with a nontargeting control siRNA. TaqMan Gene Expression Assays against the 18S rRNA were used to normalize for differences in total RNA concentration.

Higher Potency siRNA Means Higher Specificity siRNA

To examine the impact of using higher potency siRNAs at lower concentrations on siRNA specificity, we employed a global gene profiling assay using Affymetrix U133 2.0 plus arrays, which contain probes to over 47,000 human transcripts. Array analysis was performed with triplicate HeLa cell samples transfected for 24 hours with 30 nM or 3 nM Silencer Select siRNAs targeting CLTC. Controls included transfection of a nontargeting negative control siRNA at 30 nM. Statistical analysis was then performed to determine the number of genes showing changes in expression greater than 2-fold with a P value of <0.001 ("differently expressed genes," DEGs; Figure 2).


Figure 1. Potency of Ambion Silencer Select siRNAs Targeting CLTC Gene. Three Silencer Select siRNAs targeting human CLTC were reverse transfected into HeLa cells in triplicate at 5 different concentrations in 24-well plates using 1.5 uL siPORT™ NeoFX™ Transfection Agent. 24 hr post-transfection, RNA was isolated from cells using the MagMAX™-96 Total RNA Isolation Kit. cDNA was synthesized with the Applied Biosystems High Capacity cDNA Reverse Transcription Kit and was used to amplify CLTC mRNA in RT-PCR reactions with CLTC Hs00191535_m1 TaqMan Gene Expression Assay primers. Percent gene expression remaining was expressed as the relative amount of CLTC mRNA in cultures transfected with CLTC targeting siRNAs versus cells transfected with a nontargeting control siRNA. TaqMan Gene Expression Assays against the 18S rRNA were used to normalize for differences in total RNA concentration.


The array data are represented in the hierarchical clustered heat map shown in Figure 3. These data represent the differentially expressed genes and the extent of their change compared to the negative control siRNA-treated samples. At 30 nM siRNA concentration, transfection by all three siRNAs reduced expression of the target gene, CLTC, greater than 8-fold compared to the negative control siRNA-transfected samples. Transfection of Silencer Select siRNAs s475 and s476 at 30 nM resulted in differential expression of 2 genes, one being the target CLTC (Figure 3, top gene). Transfection of siRNA s477 resulted in 25 DEGs; this set included the 2 DEGs found after transfection with siRNAs s475 and s476 (Figures 2 and 3). These results demonstrate minimal off-target effects and a very high degree of target knockdown precision by these siRNAs.


Figure 3. Hierarchical Clustered Heat Map Shows Low Off-Target Effects of Ambion Silencer Select siRNA. Partek’s Genomic Suite V6.2 was used to generate a heat map of Log2 Ratios (CLTC siRNA/Negative Control #1 siRNA) for all 25 DEGs resulting from transfection of Silencer Select siRNA targeting CLTC, as described in Figure 2. Average Linkage with Euclidean Distance was used for clustering both conditions and genes.

Use Lower siRNA Concentrations for Fewer Off-target Effects

Silencer Select siRNA s477 was tested to see if using less siRNA could reduce the number of off-target differentially expressed genes without compromising the knockdown of the target gene, CLTC. In Figure 4, the genes that showed 2-fold or greater differential expression at 30 nM are depicted in blue. The genes corresponding to the 2-fold changes are listed on the X-axis and the log ratio of the expression change is plotted on the Y-axis. Transfection at 30 nM by Silencer Select siRNA s477 resulted in 25 DEGs, but transfection by the same siRNA at 3 nM final concentration resulted in 6 DEGs, a 74% reduction compared to the 30 nM siRNA transfection conditions. Despite the dramatic reduction in DEGs with the lower siRNA concentration, there was little change in the CLTC target knockdown. This experiment again shows how Silencer Select siRNAs can be used at lower concentrations to achieve equivalent functional knockdown, with the additional benefit of a dramatic increase in specificity as determined by microarray analysis.
Figure 4. Lower Transfection Concentration of Ambion Silencer Select siRNA Results in Reduction of Off-Target Effects. A profile plot was generated with Partek GS V6.2 and shows the Log2 ratios (Silencer Select siRNA s477/Negative Control siRNA) for both 3 nM (1 replicate array) and 30 nM (mean of biological triplicates) transfections performed as described in Figure 2.

Cleaner, More Consistent Cell-based Data

The new Silencer Select siRNAs, designed using the SVM-based algorithm and including specificity-enhancing chemical modifications, are highly potent and capable of producing maximal knockdown at both high (100–30 nM) and low (3 nM) concentrations. Because Silencer Select siRNAS are effective at lower concentrations, more experiments can be conducted with the same amount of siRNA as compared to previous siRNA technologies, making these new siRNAs more economical. As demonstrated by the data presented here, an even more significant benefit of using Silencer Select siRNAs at lower concentrations is that specificity can be further improved as measured by global gene profiling. A highly functional siRNA with increased specificity means better gene knockdown and fewer off-target effects—ultimately resulting in cleaner experimental results in cell-based assays.

Scientific Contributors
Susan Magdaleno, Lesslie Beauchamp, Penn Whitley • Applied Biosystems, Austin, TX