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INTEGRATEDDNATECHNOLOGIESPRODUCTSFUNCTIONAL GENOMICSLarge-Scale DsiRNAsRNA duplexes are available up to 10 grams. All duplexes are purified using RNase-free HPLC methods and come with purityand ESI-MS QC documents.Researchers planning in vivo work should be aware that potentially toxic substances can be introduced during oligonucleotidesynthesis and purification (HPLC) processes that must be removed to prevent errors or artifacts.Proper manufacturing protocols for large-scale DsiRNA synthesis include:• Adding trace EDTA in HPLC buffers to remove heavy metal cations• Using only biocompatible buffers and salts such as sodium phosphate and sodium chloride for HPLC• Maintaining an endotoxin-free environment• Options for sterile filtration and endotoxin testingThe Endotoxin ProblemThe classic endotoxin is lipopolysaccharide (LPS), a natural product present in the outer membrane of the cell wall of Gramnegative bacteria. Toxic LPS is liberated whenever these bacteria die. Its presence can cause severe inflammatory responsesand/or death of the animals. In mammals, LPS binds to serum proteins and interacts with various receptors on monocytes,macrophages, and endothelial cells. This eventually triggers cytokine production, complement activation, and coagulationcascades.IDT large-scale siRNAs are manufactured with in vivo use in mind. Protocols are employed that minimize the risk of endotoxincontamination.As an option, duplexes can be ordered as sterile filtered, endotoxin-free. Endotoxin testing will be performed to verify thatlevels do not exceed 0.1 EU/mg.Please inquire with a local sales office for a custom quote.Dicer-substrate RNAi methods were developed in a collaborative project between IDT and Drs. John Rossi and Dongho Kim at the BeckmanResearch Institute of the City of Hope National Medical Center. 1,2 IDT is licensed under patent applications jointly filed by IDT and The City ofHope Medical Center to sell research products incorporating Dicer-substrate RNAi technology.Related ProductsRNaseAlert and DNaseAlert Pg. 38Duplex Buffer Pg. 40References:1. Synthetic dsRNA Dicer-substrates enhance RNAi potency and efficacy. Kim, D.H., et al., Nat Biotechnol, 23(2): p. 222-6 (2005).2. Functional polarity is introduced by Dicer processing of short substrate RNAs. Rose, S.D., et al., Nucleic Acids Res, 33(13): p. 4140-56 (2005).©2008 Integrated DNA Technologies www.idtdna.com17
INTEGRATEDDNATECHNOLOGIESPRODUCTSFUNCTIONAL GENOMICSTriFECTaDicer-substrate RNAi kits for gene knockdownThe Trifecta kit contains three Dicer-substrate (DsiRNA) duplexes targeting a specific gene that are selected from apredesigned collection of duplexes in the DsiRNA RefSeq Library. The DsiRNA target sites were chosen by a rational designalgorithm that integrates both traditional 21-mer siRNA design rules as well as new Dicer-substrate design criteria.In addition to three target-specific screening duplexes, the Trifecta kit contains three control sequences that are needed toperform RNAi experiments, including: (1) a TYE 563-labeled transfection control DsiRNA duplex, (2) a scrambled universalnegative control DsiRNA duplex which is absent in human, mouse, and rat genomes, and (3) a positive control DsiRNA duplexwhich targets a site in the HPRT (hypoxanthine guanine phosphoribosyltransferase) gene that is common between human,mouse, and rat and is pre-validated to give >90% knockdown of HPRT when transfected at 10 nM concentration.IDT guarantees that at least two of the three screening DsiRNA duplexes in the Trifecta kit will give at least 70% knockdownof the target mRNA when used at 10 nM concentration and assayed by quantitative RT-PCR when the fluorescent transfectioncontrol duplex indicates that >90% of the cells have been transfected and the HPRT positive control works with the expectedefficiency.Trifecta Kit Contents• Three target-specific Screening DsiRNA duplexes (2 nmoles each)• Fluorescent-labeled transfection control duplex: TYE 563 (1 nmole)• HPRT-S1 DS Positive Control duplex (1 nmole)• DS Scrambled-Neg, universal negative control duplex (1 nmole)• RNase-Free Duplex Buffer (100mM KAc/30 mM HEPES pH 7.5)THESE PRODUCTS ARE NOT FOR USE IN HUMANS OR NON-HUMAN ANIMALS AND MAY NOT BE USED FOR HUMAN ORVETERINARY DIAGNOSTIC, PROPHYLACTIC OR THERAPEUTIC PURPOSES.References1. Synthetic dsRNA Dicer-substrates enhance RNAi potency and efficacy. Kim, D.H., et al., Nat Biotechnol, 23(2): p. 222-6 (2005).2. Functional polarity is introduced by Dicer processing of short substrate RNAs. Rose, S.D., et al., Nucleic Acids Res, 33(13): p. 4140-56 (2005).Technical NoteBioinformatics support personnel can assist with special design needs. Please contact a local sales office.©2008 Integrated DNA Technologies 18 www.idtdna.com
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