RNA silencing : methods and protocols

Preface ..............................................................................................................v Contributors ..................................................................................................... ix 1 Identification of Components of RNAi Pathways Using the Tandem Affinity Purification Method Mikiko C. Siomi and Haruhiko Siomi ................................................... 1 2 Separation of Drosophila RNA Silencing Complexes by Native Gel Electrophoresis John W. Pham and Erik J. Sontheimer ................................................ 11 3 Preparation and Analysis of Drosha Yoontae Lee and V. Narry Kim ........................................................... 17 4 RNA Interference in Caenorhabditis elegans Nathaniel R. Dudley and Bob Goldstein ............................................ 29 5 Down-Regulating Gene Expression by RNA Interference in Trypanosoma brucei Christine E. Clayton, Antonio M. Estévez, Claudia Hartmann, Vincent P. Alibu, Mark Field, and David Horn .............................. 39 6 Analysis of Double-Stranded RNA and Small RNAs Involved in RNA-Mediated Transcriptional Gene Silencing M. Florian Mette, Werner Aufsatz, Tatsuo Kanno, Lucia Daxinger, Philipp Rovina, Marjori Matzke, and Antonius J. M. Matzke ............................................................. 61 7 Strategies for the Design of Random siRNA Libraries and the Selection of Anti-GFP siRNAs Mouldy Sioud and Sébastien Wälchli ................................................. 83 8 Silencing Gene Expression with Dicer-Generated siRNA Pools Jason W. Myers and James E. Ferrell, Jr. ............................................ 93 9 Inhibition of Gene Expression In Vivo Using Multiplex siRNA Sung-Suk Chae and Timothy Hla ...................................................... 197 10 Gene Silencing by a DNA Vector-Based RNAi Technology Guangchao Sui and Yang Shi ............................................................ 205 11 Plasmid-Based RNA Interference: Construction of Small-Hairpin RNA Expression Vectors Scott Q. Harper and Beverly L. Davidson ........................................ 219 vii viii Contents 12 siRNA Delivery In Vivo Mouldy Sioud .................................................................................... 237 13 Peptide-Based Strategy for siRNA Delivery into Mammalian Cells Federica Simeoni, May C. Morris, Frederic Heitz, and Gilles Divita ........................................................................... 251 14 Lentiviral Vector Delivery of siRNA and shRNA Encoding Genes into Cultured and Primary Hematopoietic Cells Mingjie Li and John J. Rossi .............................................................. 261 15 Exon-Specific RNA Interference: A Tool to Determine the Functional Relevance of Proteins Encoded by Alternatively Spliced mRNAs Alicia M. Celotto, Joo-Won Lee, and Brenton R. Graveley .............. 273 16 Immunoprecipitation of MicroRNPs and Directional Cloning of MicroRNAs Elisavet Maniataki, Maria Dels Angels De Planell Saguer, and Zissimos Mourelatos .............................................................. 283 17 Detection of MicroRNAs and Assays to Monitor MicroRNA Activities In Vivo and In Vitro Marianthi Kiriakidou, Peter Nelson, Stella Lamprinaki, Anup Sharma, and Zissimos Mourelatos ...................................... 295 18 High-Throughput Analysis of MicroRNA Gene Expression Using Sensitive Probes Mouldy Sioud and Øystein Røsok ..................................................... 311 19 Modification of Human U1 snRNA for Inhibition of Gene Expression at the Level of Pre-mRNA Peng Liu, Mary L. Stover, Alexander Lichtler, and David W. Rowe ...................................................................... 321 Index ............................................................................................................ 333