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RNAi miRNA and siRNA DNAProtein • DNA RNA • RNAProtein http://www.nobelprize.org/educational/medic ine/dna/index.html mRNA • The instructions for making protein • If you can destroy the mRNA the protein won’t be made • How could you destroy only the harmful mRNA without hurting all the other RNA in the cell (the cell is making many useful proteins) HINT1: How does this relate to viruses? • Some viruses have double stranded RNA • To survive, human cells had to destroy all double stranded RNA in the cell (the normal cell doesn’t have any) • Use something that is already inside the cell! HINT 2: • Remember! Viruses make copies of their DNA/RNA inside the host cell- destroying one copy is not enough HINT 3 • Think about complementary pairs! The answer! • siRNAs- silencing RNAs • 1) Insert a double stranded piece of RNA with one piece complementary to the RNA you want to destroy • 2)The cell will destroy this siRNA, but first it will take one side (single stranded RNA) and march around the cell destroying any RNA that matches that RNA RNAi • http://www.nature.com/nrg/multimedia/rnai/ animation/index.html How can this be used for treatment of cancer? Practical side of RNAi • http://www.invitrogen.com/site/us/en/home/ References/Ambion-Tech-Support/rnaisirna/tech-notes/rnai-how-to--for-newusers.html Scientific Techniques • You have to insert a dsRNA that is less than 30 base pairs long, because 30+ base pairs makes the cell mount a much stronger antiviral response • The RNA should match exactly the gene you want to silence From Invitrogen Reagents Required for RNAi Experiments The reagents required for inducing and analyzing the RNAi effect are quite simple: • A specific dsRNA that targets a particular gene transcript to induce the RNAi pathway • An efficient dsRNA delivery system • Assays for the RNAi effect • Proper controls specific dsRNA that targets a particular gene • Must know the sequence • Can synthesize RNA chemically efficient dsRNA delivery system • Immortalized cell lines: “transfection with a lipid- or amine-based reagent is the preferred option” • Other cells: “electroporation using a specialized, gentle-on-cells buffer and optimized pulsing conditions generally results in very efficient siRNA delivery without compromising cell viability” Control Cells • Cells that the “silenced” cells can be compared to • What should you do? Assays for the RNAi effect • Need: something that monitors the mRNA levels in the cell • Want: Something that measures gene activity – Concentration of enzyme – A certain metabolic pathway This seems REALLY difficult “Ambion, in partnership with Cenix BioScience, provides expert designed, guaranteed-tosilence siRNAs to >34,000 human, mouse, and rat targets (>98% of all human, mouse, and rat genes in the RefSeq database).”
