FlexiPlate siRNA provides highly flexible RNAi screening and is available at 0.1 nmol, 0.25 nmol, and 1 nmol scales in 96-well plates, and at 0.1 nmol and 0.25 nmol scales in 384-well plates for a choice of target genes. For maximum flexibility, siRNAs can be selected and plate layout specified at the GeneGlobe Web portal. Lists of preselected siRNAs are also available for many gene families. siRNAs have been designed using HP OnGuard siRNA Design, which incorporates neural network technology, proprietary homology analysis, and advanced features, such as 3' UTR/seed region analysis, asymmetry, SNP avoidance, and interferon motif avoidance. Note: QIAGEN does not provide FlexiPlate siRNA in pools.
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). HP OnGuard siRNA Design features Feature Description References 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. 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 1. Huesken, D. et al. (2005) Design of a genome-wide siRNA library using an artificial neural network. Nat. Biotechnol. 23, 995. 2. Mukherji, M. et al. (2006) Genome-wide functional analysis of human cell-cycle regulators. Proc. Natl. Acad. Sci. 103, 14819. 3. Matveeva, O. et al. (2007) Comparison of approaches for rational siRNA design leading to a new efficient and transparent method. Nucleic Acids Res. 35, e63. 4. Krueger, U. et al. (2007) Insights into effective RNAi gained from large-scale siRNA validation screening. Oligonucleotides 17, 237. 5. Aza-Blanc, P. et al. (2003) Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. Mol. Cell 12, 627. 6. Schwarz, D.S. et al. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199. 7. Farh, K.K. et al. (2005) The widespread impact of mammalian microRNAs on mRNA repression and evolution. Science 310, 1817. 8. Grimson, A. et al. (2007) MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell 27, 91. 9. Jackson, A.L. et al. (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635. 10. Lewis, B.P., Burge, C.B., and Bartel, D.P. (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15. 11. Lim, L.P. et al. (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769. 12. Saxena, S., Jónsson, Z.O., and Dutta, A. (2003) Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells. J. Biol. Chem. 278, 44312. 13. Judge, A.D., Sood, V., Shaw, J.R., Fang, D., McClintock, K., and MacLachlan, I. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol. 23, 457. 14. Hornung, V. et al. (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med. 11, 263. 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. >
FlexiPlate siRNA allows design of RNAi screening experiments to suit specific requirements. siRNAs for any human or mouse gene can be selected as well as any positive and negative controls required. With the user-friendly Web interface, siRNAs and controls can be placed in any of the wells of a 96-well or 384-well plate, or plate layout can be selected from a range of predefined layout patterns. siRNAs are provided in 0.1 nmol, 0.25 nmol, or 1 nmol scales, enabling economical screening using low or higher siRNA amounts, as required. The 0.1 nmol, 0.25 nmol, and 1 nmol scales are available in 96-well plates. The 0.1 nmol and 0.25 nmol scales are available in 384-well plates.
The GeneGlobe Web portal makes it easy to search for siRNAs for genes of interest and to arrange plate layouts to suit screening experiments. Lists of gene names, Entrez Gene IDs, RefSeq IDs, siRNA names, or catalog numbers can be uploaded, making the process of plate ordering fast and easy. Order plates immediately or save for later changes and ordering. Easily download plate layouts for your records.
