Human ON-TARGETplus siRNA Library - Ubiquitin Conjugation Subset 3

siRNA designed and modified for greater specificity


An arrayed collection of siRNA reagents for RNAi screening, targeting human RING finger and RING-finger-like E3 ligases. ON-TARGETplus siRNA is guaranteed to silence, and is modified to reduce off-targets for fewer false positives.

The Human ON-TARGETplus Ubiquitin Conjugation Subset 3 siRNA library contains RING finger and RING finger-like E3 Ligases; the large majority of known ubiquitin ligases. RING finger E3 ligases and most of the RING finger-like 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. A20-like proteins, also included in this set, represent the newest class of E3 ligases and contain a single coordination site for zinc. This set contains siRNA for well-known E3s, such as BRCA1, MDM2, and CBL family members.

Highlights

  • Patented dual-strand ON-TARGETplus modification pattern on all siRNAs to reduce off-targets
    • Sense strand is modified to prevent interaction with RISC and favor antisense strand uptake
    • Antisense strand seed region is modified to destabilize off-target activity and enhance target specificity
  • Available as SMARTpool siRNA reagents or a Set of 4 siRNAs in 96-well plates
  • Guaranteed target gene knockdown (see Specifications tab)

Gene targets

For a complete list of target genes in this siRNA Library, please email Technical Support, or call 1-800-235-9880. International customers, please call 303-604-9499 or your local Sales Representative.

Experimental considerations

For a thorough investigation of the ubiquitin pathway, you may also consider these additional libraries:

  
HazardousNo
Shipping ConditionAmbient
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-20 C

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 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.

False phenotypes due to off-targets are alleviated by ON-TARGETplus SMARTpool reagents while target gene knockdown is maintained

False phenotypes due to off-targets are alleviated by ON-TARGETplus SMARTpool reagents while target gene knockdown is maintained

False phenotypes due to off-targets are alleviated by ON-TARGETplus SMARTpool reagents while target gene knockdown is maintained

The effect of silencing ARPC1B on cell migration was studied in a breast cancer cell line. A monolayer of cells was uniformly scraped and the rate of cell migration to close the scrape (wound healing) was evaluated. Both unmodified and ON-TARGETplus siRNA reagents induced potent target knockdown. Inconsistent phenotypes due to off-target effects (red outline), were observed for cells transfected with unmodified individual siRNAs. The unmodified SMARTpool improved the false phenotype considerably, while the ON-TARGETplus SMARTpool significantly reduced off-target effects to produce a consistent phenotype. In collaboration with Kaylene Simpson, Laura Selfors, and Joan Brugge, Harvard Medical School.


ON-TARGETplus modifications reduce the overall number of off-targets and pooling reduces them even further

ON-TARGETplus modifications reduce the overall number of off-targets and pooling reduces them even further

ON-TARGETplus modifications reduce the overall number of off-targets and pooling reduces them even further

(A) and (B) are representative examples of off-target signatures with and without application of ON-TARGETplus modifications to (A) a single siRNA and (B) a SMARTpool reagent. Green bars indicate genes with 2-fold or more reduction of expression when treated with the indicated siRNA reagent. The ON-TARGETplus modifications reduced the off-targets when compared to unmodified siRNA. Pooling of siRNA and the ON-TARGETplus modification pattern independently, and in combination, provide significant reduction in off-target gene silencing. Panel (C) represents quantitation of off-targets (down-regulated by 2-fold or more) induced by the indicated siRNA reagents targeting 10 different genes (4 siRNAs per gene or a single SMARTpool reagent). Off-targets were quantified using microarray analysis (Agilent) then compiled. Each shaded box represents the middle 50% of the data set. Horizontal line in box: Median value of the data set. Vertical bars: minimum and maximum data values.


References

  1. B. D. Parsons, A. Schindler, D. H. Evans, E. Foley, A direct phenotypic comparison of siRNA pools and multiple individual duplexes in a functional assay. PLoS One. 4(12), e8471 (2009).
  2. M. Jiang, R. Instrell, B. Saunders, H. Berven, M. Howell, Tales from an academic RNAi screening facility; FAQs. Brief Funct. Genomics. 10(4), 227-237 (2011). [doi: 10.1093/bfgp/elr016]

Citations

  1. T. Ratovitski, E. Chighladze, E. Waldron, R. R. Hirschhorn, C. A. Ross, Cysteine proteases bleomycin hydrolase and cathepsin Z mediate N-terminal proteolysis and toxicity of mutant huntingtin. J. Biol. Chem. 286(14), 12578-12589 (2011). [Human Proteases]