Human siGENOME RTF - Ubiquitin Conjugation subset 3

Ideal for labs who want to carry out siRNA screens but lack high-throughput capabilities.


A ready-to-use reverse transfection format RNAi screening library targeting human RING-finger E3 ligases. Just resuspend pre-dispensed siRNA, and add cells. Optimization plates available.

The Human siGENOME RTF Ubiquitin Conjugation 3 siRNA Library contains the majority of known ubiquitin ligases. RING finger and most RING finger-like E3 ligases form a site of interaction with E2s through a platform generated as a consequence of coordination of two zinc ions. A20-like proteins, also included in this set, contain a single coordination site for zinc. This set contains siRNA for well-known E3s, such as BRCA1, MDM2, and CBL family members.

siGENOME siRNAs are designed with the proprietary SMARTselection design algorithm for high-efficiency, guaranteed silencing. They also incorporate rational strand bias with application of ON-TARGET modifications to optimize antisense strand loading into RISC for effective target knockdown.

Highlights

  • Six ready-to-use 96-well plate sets provided for two triplicate screens
  • Pre-plated, validated RNAi controls included - reduces hands-on time for faster screening results
  • No aliquoting necessary - just resuspend, and add cells
  • SMARTpool siRNA reagents provided in clear plates at 6.25 pmol per well (50 nM final screening concentration)
  • Black or white clear-bottom plates available to support assays involving fluorescent or luminescent detection
For a diagram of the RTF Library Plate layout, see Figure 1 on the Supporting Data tab.
Well Pre-plated control Control Catalog No.
A1 siGENOME Non-targeting siRNA #2 D-001210-02
B1 siGENOME Non-targeting siRNA #3 D-001210-03
C1 siGENOME Non-targeting siRNA #4 D-001210-04
D1 siGENOME Non-targeting siRNA #5 D-001210-05
E1 siGENOME Non-targeting pool #2 D-001206-14
F1 siGENOME Cyclophilin B control pool (H, M, R) Control siRNA D-001136-01

For a diagram of the RTF Library Plate layout, see Figure 1 on the Supporting Data tab.

Experimental considerations

DharmaFECT Cell Culture Reagent (DCCR) is recommended for use with RTF libraries to dilute transfection reagents prior to use. DCCR may be purchased separately or accompanying your RTF Optimization Plates

DharmaFECT transfection reagents are highly recommended for use with RTF libraries and should be purchased separately. Refer to the DharmaFECT Cell Type Guide to find the appropriate formulation for your cell type.

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.

For a thorough investigation of the ubiquitin pathway, you may also consider our three Ubiquitin Conjugation libraries:

  
HazardousNo
Shipping ConditionAmbient
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition4 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.

Plate layout of siGENOME RTF siRNA Libraries

Plate layout of siGENOME RTF siRNA Libraries

Plate layout of siGENOME RTF siRNA Libraries

Figure 1 | Validated control siRNAs and pools are pre-dispensed into column 1 of each RTF Library plate, providing a consistent baseline for screening and assay efficiency.


Reverse Transfection Format is highly effective across cell lines

Reverse Transfection Format is highly effective across cell lines

Reverse Transfection Format is highly effective across cell lines

Figure 2 | Reverse transfection format was used to assess control gene silencing (Cyclophilin B; blue bars) and viability (yellow dots) across eight cell lines under optimized conditions. In all cases, effective target gene knockdown was achieved with low cytotoxicity.


Simple four-step protocol for screening with RTF SMARTpool siRNA Libraries

Simple four-step protocol for screening with RTF SMARTpool siRNA Libraries

Simple four-step protocol for screening with RTF SMARTpool siRNA Libraries

Figure 3 |


References

  1. Publications using RTF Libraries
  2. T. Sorkina, M. Miranda, K.R. Dionne, B.R. Hoover, N.R. Zahniser, A. Sorkin, RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis. J Neurosci. 26(31), 8195-205 (2006).
  3. P. Monteiro, D. Gilot, E. Le Ferrec, C. Rauch, D. Lagadic-Gossmann, O. Fardel, Dioxin-mediated up-regulation of aryl hydrocarbon receptor target genes is dependent on the calcium/calmodulin/CaMKIalpha pathway. Mol Pharmacol. 73(3), 769-77 (2008). Epub 2007 Dec 18.
  4. A.A. Kolokoltsov, D. Deniger, E.H. Fleming, N.J. Roberts Jr, J.M. Karpilow, R.A. Davey, Small interfering RNA profiling reveals key role of clathrin-mediated endocytosis and early endosome formation for infection by respiratory syncytial virus. J Virol. 81(14), 7786-800 (2007). Epub 2007 May 9.
  5. K.M. Hussain, K.L. Leong, M.M. Ng, J.J. Chu, The essential role of clathrin-mediated endocytosis in the infectious entry of human enterovirus 71. J Biol Chem. 286(1), 309-321 (2011). Epub 2010 Oct 18.
  6. General Screening References
  7. 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).
  8. 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. Epub 2011 Apr 28.