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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