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INTEGRATEDDNATECHNOLOGIESPRODUCTSFUNCTIONAL GENOMICSTriFECTin TM Transfection ReagentTrifectin is a proprietary cationic lipid formulation that has been optimized for delivery of IDT’s Dicer-substrate siRNAsinto a wide variety of cell types with minimal toxicity. It is equally potent for delivery of traditional 21-mer siRNAs and isalso effective in delivering other kinds of nucleic acids. In particular, Trifectin has been shown to be effective in deliveringantisense oligos (ASOs) of all kinds, including anti-miRNA ASOs. Trifectin is also effective in delivering plasmid DNAs into cells.Benefits of Trifectin include:•••Easy optimization due to low toxicity and good function across a wide range of RNA/lipid transfection ratiosEfficient transfection permitting use of lower RNA concentrationsFunctional in a wide range of cell types – see the Trifectin Technical Bulletin on IDT's website for up-to-date protocolsfor different cellsProduct0.5 mL Trifectin1 mL Trifectin5 x 1 mL TrifectinTechnical NoteContents and StorageHandle under sterile conditions. Store at +4°C. Trifectin is guaranteed to be stable for a minimum of six months whenproperly stored. Do not freeze or leave at room temperature for extended periods of time.To qualitatively assess transfection efficiency, we recommend using the dye-labeled transfection control duplexes. Toquantitatively assess transfection efficiency, we recommend using the positive control duplexes. A variety of positivecontrols are available.Basic ProtocolMix Trifectin with the siRNA or other nucleic acid, add to the cells, and incubate. Trifectin has low toxicity and it is notnecessary to remove the transfection cocktail before an extended incubation period. Use is compatible with bothforward and reverse transfection and in manual or robotic systems.Related ProductDsiRNA Screening Duplexes Pg. 15©2008 Integrated DNA Technologies www.idtdna.com23
INTEGRATEDDNATECHNOLOGIESPRODUCTSFUNCTIONAL GENOMICSDNA-Directed RNA Interference (ddRNAi)RNA interference can be achieved using RNA made enzymatically by transcription from DNA templates. This method has beencalled DNA-Directed RNAi, or ddRNAi. In its simplest form, ddRNAi can be done in vitro where a T7 or any other convenientpromoter/polymerase combination is used to make RNA from template DNA oligos 1 . These products are heterogeneous andcan sometimes result in interferon induction or other related problems 2 . However, this approach does offer an inexpensiveway to make RNA.DNA oligos can be cloned into a variety of vectors, ranging from expression plasmids to lentiviral vectors. These constructscan be introduced into cells or animals. The cloned vectors will express the dsRNA molecules and initiate an RNAi response inthe transfected or infected cells. This method can deliver long-term instead of transient RNAi. DNA oligos can be cloned withthe sense and antisense stands in tandem 3 or in hairpin configuration, which mimics naturally occurring miRNAs. Expressedhairpins, or short-hairpin RNAs (shRNAs), are the currently favored design for the ddRNAi approach, and the construction andsystematic use of large-scale libraries of this kind for functional genomics screens are showing great promise 4, 5, 6 .ddRNAi oligos are available normalized to 4 nmoles in tubes or plates.Normally IDT recommends the use of purified oligos when cloning DNA sequences over 40 bases long. However, the hairpinpresent in the shRNA cloning oligos makes purification difficult. Further, purification is not feasible for large-volume shRNAcloning projects, where many thousands of oligos are needed quickly at low cost. IDT has developed a synthesis scale thatresults in an ultra-high quality product. This process has been optimized for manufacturing oligos in the 60-200 base range.Product identity is confirmed by LC MS spectrometry. These oligos are intended for use without additional purification.IDT is licensed under patents owned by Benitec Ltd. to sell oligos for use in ddRNAi applications.Technical NotePlease visit www.idtdna.com for information on making an shRNA expression plasmid.References:1. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Donze, O., and Picard, D. Nucleic Acids Res., 30:e46 (2002).2. Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Kim, D.-H., Longo, M., Han, Y., Lundberg, P., Cantin, E., and Rossi, J.J. NatureBiotechnology, 22:321-325 (2003).3. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Lee, N.S., Dohjima, T., Bauer, G., Li, H., Li, M.-J., Ehsani, A.,Salvaterra, P., and Rossi, J. Nature Biotechnology, 19:500-505 (2002)4. An approach to genomewide screens of expressed small interfering RNAs in mammalian cells. Zheng, L., Liu, J., Batalov, S., Zhou, D., Orth, A., Ding, S., andSchultz, P.G. Proc. Natl. Acad. Sci., USA, 101:135-140(2004).5. A resource for large-scale RNA-interference-based screen in mammals. Paddison, P.J., Silva, J.M., Conklin, D.S., Schlabach, M., Li, M., Aruleba, S., Balija, V.,O’Shaughnessy, A., Gnoj, L., Scobie, K., Chang, K., Westbrook, T., Cleary, M., Sachidanandam, R., McCombie, W.R., Elledge, S.J., and Hannon, G.J. Nature, 428:427-431(2004).6. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Berns, K., Hijmans, E.M., Mullenders, J., Brummelkamp, T.R., Velds,A., Heimerikx, M., Kerkhoven, R.M., Madiredjo, M., Nijkamp, W., Weigelt, B., Agami, R., Ge, W., Cavet, G., Linsley, P.S., Beigersbergen, R.L., and Bernards, R. Nature,428:431-7(2004).©2008 Integrated DNA Technologies 24 www.idtdna.com
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