The Mouse siGENOME Ubiquitin Conjugation 3 siRNA library targets RING finger and RING finger-like E3 ligases. These E3s form a site of interaction with E2s through a platform generated as a consequence of coordination of two zinc ions. U-box E3s conform like RING-finger proteins except that they do so through salt bridges and other interactions that do not involve coordination of zinc. This set contains A20-like proteins, which contain a single coordination site for zinc, and siRNA for well-known E3s,such as BRCA1, MDM2, and CBL family members.
siGENOME siRNA reagents are globally recognized and trusted for highly effective performance in RNAi screening applications. Our expertise in siRNA design, experimental optimization, and siRNA screening strategies provide end-to-end support of your screening efforts.
- Guaranteed target knockdown (see Specifications tab)
- Antisense strand loading into RISC ensured by thermodynamic analysis and selective application of a sense strand-blocking modification (ON-TARGET)
- Available as SMARTpool or Set of 4 siRNA reagents arrayed in 96-well plates
For a complete list of target genes in this siRNA Library, please contact Technical Support or your local Sales Representative.
For a thorough investigation of the ubiquitin pathway, you may also consider these additional libraries:
Our siRNA knockdown guarantee
siGENOME and ON-TARGETplus siRNA reagents (SMARTpool and three of four individual siRNAs) are guaranteed to silence target gene expression by at least 75% at the mRNA level when demonstrated to have been used under optimal delivery conditions (confirmed using validated positive control and measured at the mRNA level 24 to 48 hours after transfection using 100 nM siRNA).
Note: Most siGENOME and ON-TARGETplus siRNA products are highly functional at 5 to 25 nM working concentration.
Screening with siGENOME SMARTpool siRNA reagents provide effective target silencing
Effective silencing achieved for 440 kinase genes when screened with SMARTpool siRNA reagents in two cell lines. Under screening conditions these overall results provide validation of siRNA design and SMARTpool technology for effective target knockdown. HeLa and MCF10a cells were transfected with 50 nM of each siRNA using DharmaFECT 1 transfection reagent.
Target mRNA levels were determined by branched DNA assay (Panomics Quantigene® Reagent System). The pie chart represents the summary of silencing achieved for the entire data set (880 total data points). The bar graph is a representative sample of genes assayed.
Unnecessary sense-strand inactivation can increase off-target activity
Unmodified and sense strand-inactivated siRNAs were used to target five genes. In four cases, off-targets were increased due to enhanced RISC loading of the antisense strand when the sense strand was modified. The unmodified siRNAs have natural guide-strand loading characteristics. All siRNAs had comparable silencing potency. Data shown represents genes down-regulated by twofold or more. HEK293 cells were transfected with 100 nM siRNA using 0.2 μL of DharmaFECT 1. Data was analyzed at 24 hours by genomewide microarray analysis (Agilent).
Why not modify ALL siGENOME siRNAs to ensure proper strand loading? It has been demonstrated by Dharmacon scientists1 and others2 that forcing antisense (guide) strand entry into RISC may actually INCREASE off-targets due to increased loading of the guide strand and resulting off-target activity by its seed region. siGENOME siRNAs are designed with thermodynamic properties to naturally facilitate guide strand entry to RISC, which has been demonstrated to correlate with functionality1. However, in cases where a high-scoring siGENOME siRNA does not possess ideal strand-loading characteristics, a sense (passenger) strand-inhibiting chemical modification (ON-TARGET) is utilized to promote guide strand entry. Approximately 20% of siGENOME siRNAs carry the ON-TARGET modification.
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- M. Jiang et al., Tales from an academic RNAi screening facility; FAQs. Brief Funct Genomics. 10(4), 227-237 (Epub 28 April 2011, July 2011). [doi: 10.1093/bfgp/elr016]
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- J. A. Smith et al., Genome-wide siRNA screen identifies SMCX, EP400, and Brd4 as E2-dependent regulators of human papillomavirus oncogene expression. Proc. Natl. Acad. Sci. U S A. 107(8), 3752-3757. (Epub 2 February 2010, 23 February 2010).
- G. Hu et al., A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal. Genes Dev. 23(7), 837-848 (1 April 2009).
- R. D. Paulsen et al., A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability. Mol Cell. 35(2), 228-239 (31 July 2009).
- M. Tsui et al., An intermittent live cell imaging screen for siRNA enhancers and suppressors of a kinesin-5 inhibitor. PLoS One. 4(10), e7339 (5 October 2009).
- A. L. Brass et al., Identification of host proteins required for HIV infection through a functional genomic screen. Science. 319(5865), 921-926. (Epub 10 January 2008, 15 February 2008).
- C. Swanton et al., Regulators of mitotic arrest and ceramide metabolism are determinants of sensitivity to paclitaxel and other chemotherapeutic drugs. Cancer Cell. 11(6), 498-512 (11 June 2007).
- A. W. Whitehurst et al., Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Nature. 446(7137), 815-819 (12 April 2007).