RNA 干扰技术PPT.ppt

RNA interference technology; Content;Section 1 Background and ConceptsPart 1 Background;Part 1 Background;Part 1 Background;Part 1 Background;Part 1 Background;Part 1 Background;Part 1 Background;Section 1 Background and ConceptsPart 2 Concepts; What is RNA Interference?;What is RNA Interference?;;Concepts;Concepts;Concepts;Concepts;Concepts;Concepts;Concepts;Concepts;siRNA and antisense oligonucleotides ;siRNA and antisense oligonucleotides;siRNA and antisense oligonucleotides;Section 2 The mechanism of RNAi;The mechanism of RNAi;Biochemical Mechanism of RNAi;Biochemical Mechanism of RNAi;Section 3 ; siRNA Experimental Needs;siRNA Experimental Needs;Part 1: siRNA design;Part 1:siRNA design;Part 1:shRNA design;Part 2:Methods of Producing siRNAs;Chemically Synthesized siRNAs;Method 1:Chemical Synthesis;;Method 2: In vitro Transcription;Method for In Vitro Transcription ;Synthesized vs. In Vitro Transcribed;Method 3:RNase III/Dicer Digestion(Cocktail method);Method for RNaseIII Digestion;Validation of RNaseIII Digestion;RNase III Generated Cocktails;Expressing siRNAs in vivo (Method 4-6);Expressing siRNAs In Vivo;Expressing siRNAs In Vivo;Expressing siRNAs in vivo (Method 4-6);Expressing siRNAs in vivo (Method 4-6);siRNA Plasmid Expression Vectors;Method 4: Plasmid vectors;Method 4:Plasmid vectors;In Vivo Expression of siRNAs;Transient Expression Studies (Use of transient selection);Stable Down Regulation;Stable Down Regulation;Methods 5: SiRNA expression cassettes(SEC);Method 5 siRNA Expression Cassettes;siRNA Expression Cassettes;Use of siRNA Expression Cassettes;Method 6: Viral vectors;siRNA Viral Expression Vector;Part 3 Methods for Inducing RNAi in Mammalian Cells;Part 3:Delivery of siRNAs and shRNAs;Delivery of siRNAs;Delivery of siRNAs and shRNAs;Transfection of siRNAs;Overview of Optimization;Transfection Reagent Choice;Transfection of Adherent Cell Lines;Optimizing Transfection;Optimizing Transfection;Optimizing Transfection;Optimizing