05096nam 2200613Ia 450 991014455760332120170814190115.01-280-51971-197866105197123-527-60439-13-527-60437-5(CKB)1000000000377164(EBL)482269(OCoLC)68617526(SSID)ssj0000238803(PQKBManifestationID)11228463(PQKBTitleCode)TC0000238803(PQKBWorkID)10239246(PQKB)10399275(MiAaPQ)EBC482269(EXLCZ)99100000000037716420040722d2005 uy 0engur|n|---|||||txtccrRNA interference in practice[electronic resource] /Ute SchepersWeinheim Wiley-VCH20051 online resource (338 p.)Description based upon print version of record.3-527-31020-7 Includes bibliographical references and index.RNA Interference in Practice; Preface; Contents; 1 Introduction: RNA interference, the ""Breakthrough of the Year 2002""; 1.1 RNAi as a Tool for Functional Genomics; 1.2 Mechanism of RNAi; 1.3 Dicer - the Initiator to "Dice" the dsRNA?; 1.4 miRNAs versus siRNAs: Two Classes of Small RNAs Using the RNAi Pathway?; 1.5 RISC - the Effector to "Slice" the mRNA?; 1.6 Are RNA-Dependent RNA Polymerases (RdRps) Responsible for the Catalytic Nature of RNAi?; 1.7 Is RNAi Involved in the Regulation of Gene Expression?; 1.8 RNAi in Mammals; 1.9 Practical Approaches; 1.10 References2 RNAi in Caenorhabditis elegans2.1 Introduction; 2.2 Application of RNAi in C. elegans; 2.3 Target Sequence Evaluation; 2.4 dsRNA Synthesis; 2.4.2 Generation of the DNA Template; 2.4.2.1 Plasmid Templates; 2.4.2.2 DNA Templates Derived by PCR/RT-PCR; 2.4.2 In-vitro dsRNA Transcription; 2.5 Delivery of dsRNA; 2.5.1 General Information on the C. elegans Anatomy; 2.5.2 C. elegans Strains for Silencing; 2.5.3 Culturing the Worms; 2.5.4 Microinjection Protocol; 2.5.5 Soaking Protocol; 2.5.6 RNAi Feeding Protocol; 2.5.7 DNA Templates for dsRNA Expression in Feeding E. coli2.5.8 DNA Templates for Hairpin RNA Expression2.5.8.1 C. elegans Promoters; 2.5.8.2 Inverted Repeat Constructs; 2.6 Mounting Animals for Microscopy; 2.7 Genome Wide Screens; 2.7.1 C. elegans RNAi Library; 2.8 Selected Literature on C. elegans Research; 2.9 Useful C. elegans webpages; 2.10 References; 3 RNAi in Drosophila; 3.1 Introduction; 3.2 Application of RNAi in Drosophila; 3.2.1 dsRNA from Linear DNA Templates; 3.2.2 dsRNA from Inverted Repeat DNA; 3.2.3 Inducible Expression in Drosophila Cell Lines; 3.2.4 Limitations; 3.3 dsRNA Synthesis; 3.3.1 In-vitro dsRNA Transcription3.3.2 Inverted Repeat DNA3.4 Injections; 3.4.1 Injection Services; 3.4.2 Injection Method; 3.4.3 DsRNA or Inverted Repeat DNA Preparation; 3.4.4 Embryo Collection; 3.5 Cell Lines; 3.6 Protocols; 3.6.1 Thawing and Maintenance of S2 Cells; 3.6.2 Freezing Protocol; 3.7 RNAi in S2 Cells; 3.7.1 dsRNA Transfection Using the Calcium Phosphate Method; 3.7.2 dsRNA Soaking of S2 Cells; 3.8 High-Throughput Screens; 3.8.1 Drosophila RNAi Library; 3.9 Useful Webpages for Drosophila Research; 3.10 Books and Literature on Drosophila; 3.11 References; 4 RNAi in Mammals; 4.1 Introduction4.2 Transient RNAi in Cell Culture4.2.1 Chemical Synthesis and Modifications of siRNAs; 4.2.1.1 Advantages; 4.2.1.2 Limitations; 4.2.2 Custom Synthesis of siRNA Oligos; 4.2.3 siRNA Design Rules; 4.2.3.1 siRNA Strand Bias and Off-Gene Targeting; 4.2.3.2 Improvements in siRNA Stability; 4.2.3.3 siRNA Design: Novel Modifications of the "Tuschl Rules"; 4.2.3.4 Homology Search by BLAST, FASTA, or Smith-Waterman Algorithm; 4.2.3.5 Troubleshooting; 4.2.3.6 siRNA Design Programs and Algorithms; 4.2.3.7 Preparation of siRNA Duplexes; 4.2.4 Enzymatic Synthesis of siRNAs4.2.4.1 Designing DNA OligonucleotidesThis hands-on guide to RNA interference brings the power of targeted gene silencing to any laboratory with the basic equipment for handling nucleic acids.In easy-to-follow, step-by-step protocols you will learn* how RNAi works in worms, flies and mammals,* how to design the most efficient RNAi constructs,* how to achieve transient, stable and conditional RNAi in cell cultures,* how to determine the efficiency of an RNAi experiment,* and how to use RNAi for gene therapy.All the protocols have been thoroughly tested in the author's own laboratory, and she provides exaRNA editingGenetic transcriptionElectronic books.RNA editing.Genetic transcription.572.8845Schepers Ute978682MiAaPQMiAaPQMiAaPQBOOK9910144557603321RNA interference in practice2230633UNINA