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Overview: RNA interference (RNAi) by double stranded RNA (dsRNAs) molecules of approximately 20-25 nucleotides termed short interfering (siRNAs) is a powerful method for preventing the expression of a particular gene. The dsRNA dominantly silences gene expression in a sequence-specific manner by causing the corresponding endogenous mRNA to be degraded. The technique was first developed in Caenorhabditis elegans, and was rapidly applied to a wide range of organisms. Methods for expressing siRNAs in cells in culture and in vivo using viral vectors, and for transfecting cells with synthetic siRNAs, have been developed and are being used to establish the functions of specific proteins in various cell types and organisms. For example, chemically synthesized or in vitro transcribed siRNAs can be transfected into cells, injected into mice, or introduced into plants. siRNAs can also be expressed endogenously from siRNA expression vectors or PCR products in cells or in transgenic animals.
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Protocols
  • dsRNA Synthesis Protocol (Drosophila RNAi Screening Center at Harvard Medical School)
    Generating dsRNA by in vitro transcription of a PCR generated DNA template containing the T7 promoter sequence on both ends.
    http://flyrnai.org/RNAi_FAQ_dsrna_syn.html
    Added: Wed May 12 2004, Hits: 2557, Reviews: 0  Write review   Cached
  • The siRNA User Guide (Tom Tuschl, Rockefeller University )
    Offers guidance and tips on the following topics: 1) Selection of siRNA duplexes from the target mRNA sequence; 2)Preparation of siRNA duplexes; 3)Useful siRNA reagent combinations; 4)Transfection of siRNA duplexes.
    http://www.rockefeller.edu/labheads/tuschl/sirna.h...
    Added: Fri Jun 20 2003, Hits: 6426, Reviews: 1 Read reviews  Write review   Cached
  • Bacteria-Mediated (Feeding) RNAi Protocol (Schedl Lab, Washington University School of Medicine in Saint Louis)
    Feed worms with bacterially expressed dsRNAs for RNAi.
    http://www.genetics.wustl.edu/tslab/Protocols/Feed...
    Added: Sun Apr 29 2007, Hits: 343, Reviews: 0  Write review   Cached
  • dsRNAi Experiments in Drosophila Cell (Laboratory of Jack Dixon, UCSD)
    RNAi takes advantage of the unique ability of double-stranded RNA (dsRNA) molecules to induce posttranscriptional gene silencing in a highly specific manner. This silencing is efficacious and long-lived, as it is passed to subsequent generations in insect cell culture. To date, all Drosophila cell lines tested (S2, KC, BG2-C6, and Shi) respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested (more than 15) have been effective at silencing the target gene.
    http://cmm.ucsd.edu:16080/Lab_Pages/Dixon/01/RNAiE...
    Added: Fri May 09 2003, Hits: 844, Reviews: 0  Write review   Cached
  • Lentiviral RNAi Protocol (Science Gateway)
    Long DNA oligos containing the target sequence are cloned into LentiLox 3.7 (see protocol). Once clones have been isolated, virus is produced by transfecting 293 cells and collecting supernatant. This supernatant is then used to infect cells of interest directly, or concentrated for use in embryo infections.
    http://www.sciencegateway.org/protocols/lentivirus...
    Added: Fri Aug 04 2006, Hits: 1303, Reviews: 0  Write review   Cached
  • RNA Interference (RNAi) in Cultured Cells (Drosophila RNAi Screening Center at Harvard Medical School)
    The protocols listed here are for Drosophila cells in 6 well plates and our pre-aliquoted 384 well plates. RNAi experiments may be done in other size plates, just scale up or down.
    http://flyrnai.org/RNAi_FAQ_rnai_on_cc.html
    Added: Wed May 12 2004, Hits: 2793, Reviews: 0  Write review   Cached
  • RNA Interference (RNAi) Protocol (Forler D, et al.)
    This method uses the Promega Ribomax Large Scale RNA Production System T7 to produce dsRNAs and RNAi is conducted in Drosophila Schneider cells.
    http://www.mann.embl-heidelberg.de/GroupPages/Page...
    Added: Thu May 08 2003, Hits: 3328, Reviews: 0  Write review   Cached
  • RNA Interference (RNAi) Resources (Ambion)
    If you are new to RNAi, here you can find some good stuff related to RNAi, such as the mechanism of RNAi, how to design SiRNA.
    http://www.ambion.com/techlib/resources/RNAi/
    Added: Thu May 08 2003, Hits: 3319, Reviews: 0  Write review   Cached
  • RNAi-based conditional gene knockdown in mice using a U6 promoter driven vector (Vivek Shukla, Xavier Coumoul, Chu-Xia Deng, Genetics of Development and Disease Branch, 10/9N105, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health)
    RNA interference (RNAi) is a powerful tool widely used for studying gene function in a number of species. An approach that allows conditional expression of a polymerase III promoter based small hairpin RNA (shRNA) in mice using the Cre-LoxP system has been developed. This approach uses a U6 promoter, which is inactive due to the presence of a ploxPneo cassette in the promoter; this promoter can be activated after excision of the neo gene in transgenic mice that express a Cre recombinase transgene. As a proof of principle, we have previously knocked down over 95% of Fgfr2 transcripts in mouse germlines, leading to embryonic lethality, while restricting the knockdown to the progress zone of the limb results in live animals with malformation of digits of both the forelimbs and hindlimbs. We now provide a detailed protocol, including a simplified single-step cloning procedure for vector construction. This method provides a fast yet efficient way to decipher gene functions in vivo in a tissue specific manner.
    http://www.biolsci.org/v03p0091.htm
    Added: Sat Mar 29 2008, Hits: 130, Reviews: 0  Write review  
  • siRNA Protocols (Mellor Laboratory Bristol)
    This page decribes siRNA design, in vitro synthesis, annealing and transfection.
    http://www.bch.bris.ac.uk/staff/Mellor/docs/protoc...
    Added: Sun Jun 05 2005, Hits: 1262, Reviews: 0  Write review   Cached
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