The Human ON-TARGETplus Ubiquitin Conjugation Subset 1 siRNA library serves as a preliminary evaluation of the potential role of the ubiquitin system in processes under study. Assessment of effects of E1 and E2 enzymes will provide a reasonable idea as to whether components of the ubiquitin system are involved.
Also included in this set are the HECT domain E3 ligases. Members of this relatively small family of E3s differ from the large majority of known E3s (RING finger and RING finger-like) in functioning as catalytic intermediates in ubiquitin conjugation. Members of this family, particularly Nedd4 and related WW-domain containing E3s, and E6-AP are implicated in the ubiquitylation of many different proteins. Some lead to proteasomal degradation and others to non-proteasomal ubiquitylation.
- Includes Cullin, E1, E2, HECT E3 Ligases
- 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)
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.
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
(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.
- 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).
- 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. doi: 10.1093/bfgp/elr016 (2011).
- 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]
Concentrated buffer solution recommended for resuspension and long-term storage of any short, double-strand, or single-strand synthetic RNA molecule. Dilute with RNase-free water prior to use.
Molecular grade water for dilution of 5x siRNA Buffer or resuspension of RNA. RNase-free to prevent degradation of RNA reagents and oligonucleotides.
An aliquot of each of the four DharmaFECT formulations for siRNA/microRNA transfection optimization studies