The Human siGENOME Ubiquitin Enzymes Library combines gene targets from four of our ubiqutin-related libraries into one comprehensive collection for investigation of regulators of the ubiqutination pathway:
- Deubiquitinating Enzymes
- Ubiquitin Conjugation Subset 1 - Cullins, E1, E2, HECT E3 Ligases
- Ubiquitin Conjugation Subset 2 - F-box, SOCS box E3 Ligases
- Ubiqutin Conjugation Subset 3 - RING finger and RING finger-like E3 Ligases
- Antisense strand loading into RISC ensured by thermodynamic analysis and selective application of a sense strand-blocking modification (ON-TARGET)
- Available as SMARTpool siRNA reagents or a Set of 4 siRNAs in 384-well plates
- Guaranteed target gene knockdown (see Specifications tab)
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.
For a complete list of target genes in this siRNA Library, please contact Technical Support or your local Sales Representative.
|Storage Conditions||-20 C|
|Stability at Recommended Storage Conditions||At least 12 months|
Screening with siGENOME SMARTpool siRNA reagents provides 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.