1. Is the Silencer Select siRNA a chemically modified siRNA? If so, what is the modification?
Yes; the exact modification is proprietary, and it reduces overall off target effects by up to 90% without compromising potency. The modification also exaggerates strand bias, which correlates with better knockdown. Furthermore, we have shown that the modification produces more concordant phenotypes in cell-based assays as compared to unmodified siRNA.
2. How does the 100% guarantee work?
Life Technologies guarantees that when you purchase two Silencer Select Pre-designed siRNA to the same target, then those two siRNAs will silence the target mRNA by 70% or more. To qualify for the guarantee, siRNA's must have been transfected at ≥5 nM and mRNA levels detected 48h post transfection. Real-Time RT-PCR is recommended but not required for this application. Customers must also show > 80% knockdown with a positive control siRNA to demonstrate transfection efficiency. If the guaranteed level of knockdown is not observed and an appropriate positive control is successful, a onetime replacement of up to two new Silencer Select siRNA designs will be provided free of charge. This guarantee does not extend to any replacement product.
PRODUCT REPLACEMENT AS SET FORTH ABOVE IS LIFE TECHNOLOGIES’ SOLE OBLIGATION AND CUSTOMER’S SOLE AND EXCLUSIVE REMEDY WITH RESPECT TO THE GUARANTEE GIVEN ABOVE. To arrange a replacement, contact Technical Services. Please be ready to supply data indicating transfection efficiency and measurement of mRNA knockdown. Contact Technical Support if you require such a replacement.
3. What is the status of your current Silencer siRNAs? Can we still order those?
Silencer siRNAs are still available with the same guarantee and pricing.
4. What if you don’t have a Silencer Select siRNA for my target gene?
If we do not currently have a siRNA sequence for your target gene, we can apply the same algorithm used for our Silencer Select Pre-designed siRNAs to the design of a Silencer Select siRNA for your target gene. That siRNA will contain the same modifications as found in our other Silencer Select siRNAs. (Note that these Custom Designed Silencer Select siRNAs are will be described on your order as “Silencer Select Pre-designed siRNAs, Non-inventoried”). Please contact our Technical Support Group to request Custom Designed Silencer Select siRNAs.
5. Are your Silencer Select positive control sequences different?
The Silencer Select GAPDH Positive Control siRNA sequence has not changed; it has the same sequence as AM4624 (Silencer GAPDH siRNA) but with the Silencer Select modifications.
6. Are these modified positive control sequences more potent?
In our experiments, the Silencer Select GAPDH Positive Control siRNA has similar potency to our Silencer GAPDH siRNA.
7. Are the Silencer Select Negative Control sequences different?
The Silencer Select Negative Controls #1 and #2 are entirely new sequences generated by using more stringent specificity filters. Furthermore, these have been array validated and have been tested in multi-parametric cell based assays and shown to have minimal effects on gene expression or cell phenotype.
8. How are the Silencer Select siRNAs improved over Silencer siRNAs?
These siRNA sequences are generated with a new algorithm. This algorithm was developed by Ambion’s bioinformatics group using a support vector machine (SVM) approach. The SVM approach takes a classification approach rather than a linear regression approach, and results in the ability to differentiate “hyperfunctional” versus “acceptable” siRNAs.
Specificity is enhanced by including bioinformatic filters that remove siRNAs with too few mismatches to unintended targets--those that are potentially toxic, those that match naturally occurring miRNA seed regions and those with antiviral response motifs. Also, siRNAs are ranked by frequency of potential siRNA seed region matches in the 3’UTR of off-target transcripts.
Compared to Silencer siRNA Designs, Silencer Select Designs elicit > 80% target mRNA knockdown, 28% more often.
Last but not least, Silencer Select siRNAs are modified to enhance specificity. We have shown that the modifications result in reduction of off-target effects by up to 90%, leading to more consistent, reliable phenotypic data from gene silencing experiments.
9. How does Silencer Select performance compare to your competitors?
Silencer Select siRNAs offer market-leading potency and efficacy with less off-target effects as assessed by expression analysis and cell-based phenotypic assays.
10. What are the recommended transfection amounts?
We have seen no differences in the transfection efficiency of Silencer Select siRNAs compared to unmodified siRNAs. However, Silencer Select siRNAs are 5–100 fold more potent than other siRNAs, so recommended transfection amounts are about 5–10 fold less than recommended for Silencer siRNAs (depending upon cell type used). For instance, most customers used Silencer siRNAs at ~30 nM in HeLa cells. Silencer Select siRNAs can readily be used at 3–5 nM in HeLa cells without compromising knockdown levels.
11. Are all of the Silencer Select Pre-designed and Validated siRNA sequences still disclosed upon purchase?
Yes, but the site and identity of the modification will not be disclosed.
12. How are Silencer Select siRNAs designed? Do they target just a single transcript RefSeq accession number?
Silencer Select siRNAs are designed to target all known transcripts associated with a particular gene. Ambion uses the RefSeq database (NCBI) as the transcriptome information source.
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