FlexiTube siRNA comes with a one-time-only replacement offer. If several FlexiTube siRNAs for the same target gene are ordered and at least 2 of them do not provide ≥70% target gene knockdown, QIAGEN will provide 2 additional siRNAs free of charge, once only. You will be asked to provide supporting data, demonstrating that the siRNA failed to knock down the target gene by at least 70% at the mRNA level under appropriate transfection conditions. Supporting data should include transfection efficiency data, quantitative silencing data, and data showing ≥70% knockdown of a positive control. This offer is valid for up to 6 months after the date of delivery.
Thousands of experimentally verified siRNAs
Thousands of our human siRNAs have been experimentally verified by real-time RT-PCR analysis and shown to provide at least 70% knockdown. For these verified siRNAs, experimental details and information about the level of knockdown achieved are provided in GeneGlobe. These experimentally verified siRNAs are the result of the world's largest siRNA validation project, which was carried out by QIAGEN scientists. To find out more about this project, consult the publication Krueger, U. et al. (2007) Insights into Effective RNAi Gained from Large-Scale siRNA Validation Screening. Oligonucleotides 17, 237.
Cutting-edge siRNA design
Advances in the siRNA design process ensure that QIAGEN's highly innovative and sophisticated HP OnGuard siRNA Design delivers potent and specific siRNA. siRNAs are designed using neural-network technology based on an extremely large set of data from RNAi experiments. siRNA design is then checked for homology to all other sequences of the genome using an up-to-date, nonredundant sequence database and a proprietary homology analysis tool. HP OnGuard siRNA Design incorporates many unique and advanced features (see table).
|Neural-network technology ||siRNA design uses the BioPredsi neural-network, which is based on an extremely large RNAi data set. ||1-3|
|The world's largest siRNA validation project ||The design process was reinforced and improved by data from this project, in which QIAGEN scientists proved the effectiveness of thousands of siRNAs, were used to . A large number of druggable genome siRNAs have been proven to provide at least 70% knockdown during this project. ||4|
|Homology analysis ||Analysis uses a proprietary tool and an up-to-date, nonredundant sequence database. |
|Affymetrix GeneChip analysis ||Genomewide analysis enabled development of siRNA design improvements that minimize off-target effects. |
|Up-to-date siRNA target sequences ||Current data from NCBI databases ensure accurate design. |
|Asymmetry ||siRNAs are designed with unequal stabilities of the base pairs at the 5' ends. This enables the antisense strand, which is less tightly bound at its 5' end, to enter RISC, while the sense strand is degraded. Asymmetry produces highly functional siRNAs and reduces the risk of off-target effects caused by the incorrect strand entering RISC. ||5, 6|
|3' UTR/seed region analysis ||Analysis uses intelligently weighted, multi-parameter searches for matches of the seed region of the siRNA antisense strand with the 3' untranslated region of unintended mRNA targets (see text for further explanation). ||7-12|
|SNP avoidance ||The RefSNP database is used to exclude siRNAs that span single nucleotide polymorphisms (SNPs). This increases siRNA potency, as an siRNA spanning a SNP will vary in its effectiveness. |
|Interferon motif avoidance ||siRNAs are screened for multiple sequence motifs known to result in an interferon response. siRNAs with such motifs are rejected. ||13, 14|
3' UTR/seed region analysis
Several studies have shown that off-target effects may be caused by matches of the seed region of the siRNA antisense strand with the 3' untranslated region of unintended mRNA targets (see table). The seed region comprises 6 nucleotides in positions 2–7 of the antisense siRNA strand of the siRNA duplex. Matches such as these can contribute to downregulation of unintended targets due to the siRNA mimicking the action of an miRNA. siRNA designed at QIAGEN is analyzed for 3' UTR/seed region complementarity using a proprietary set of 3' UTR sequences derived from the human, rat, and mouse RefSeq databases. Each siRNA is aligned against these sequences to check for any homology that could contribute to miRNA-like, off-target effects.
Matches of 6 out of 6 nucleotides of the siRNA seed region with an unrelated target 3' UTR sequence are common and it is not necessary or practical to eliminate siRNAs showing such matches. More rarely, seed region matches in combination with 10 or more bases of additional homology are observed in an siRNA sequence. Such homologies have greater potential to result in off-target effects, and where possible these siRNAs are rejected in favor of others with less significant homology to unintended target genes.
For some targets, it is not possible to select siRNAs that do not show any such homologies. In these cases, EntrezGene IDs of the unrelated genes that could be unintended targets of the siRNA are provided at GeneGlobe. Observation of this type of homology does not necessarily mean that these genes will be affected by the siRNA. However, they can be considered potential unintended targets for follow up analysis, if warranted.