RNA Silencing: Methods and Protocols

ISBN-13: 9781617375682 / Angielski / Miękka / 2010 / 338 str.

Gordon Carmichael

Opis

A collection of readily reproducible methods for the design, preparation, and use of RNAs for silencing gene expression in cells and organisms. The techniques range widely and include methods addressing the biochemical aspects of the silencing machinery, RNA silencing in non-mammalian organisms, and the in vivo delivery of siRNAs and silencing vectors. There are also techniques for designing, preparing, and using RNAs to silence gene expression, for fine-tuning regulation by targeting specific isoforms of a given gene, and for the study and use of microRNAs. The protocols follow the successful Methods in Molecular Biology series format, each offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls."

Recenzje

From the reviews:

"RNA Silencing: Methods and Protocols is a comprehensive and user-friendly reference and worth recommending. Researchers who are interested in using siRNA as a major method to do gene knockdown experiments in vitro to study gene function instead of using time-consuming gene targeting in mice and those who are interested in studying the functions of genes in the development of cell defense against viruses will find this book an excellent resource." (Weimin He, In Vitro Cellular & Developmental Biology - Animal, Vol. 42, May and June, 2006)

Spis treści

Identification of Components of RNAi Pathways Using the Tandem Affinity Purification Method Mikiko C. Siomi and Haruhiko Siomi Separation of Drosophila RNA Silencing Complexes by Native Gel Electrophoresis John W. Pham and Erik J. Sontheimer Preparation and Analysis of Drosha Yoontae Lee and V. Narry Kim RNA Interference in Caenorhabditis elegans Nathaniel R. Dudley and Bob Goldstein 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 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 Strategies for the Design of Random siRNA Libraries and the Selection of Anti-GFP siRNAs Mouldy Sioud and Sébastien Wälchli Silencing Gene Expression with Dicer-Generated siRNA Pools Jason W. Myers and James E. Ferrell, Jr. Inhibition of Gene Expression In Vivo Using Multiplex siRNA Sung-Suk Chae and Timothy Hla Gene Silencing by a DNA Vector-Based RNAi Technology Guangchao Sui and Yang Shi Plasmid-Based RNA Interference: Construction of Small-Hairpin RNA Expression Vectors Scott Q. Harper and Beverly L. Davidson siRNA Delivery In Vivo Mouldy Sioud Peptide-Based Strategy for siRNA Delivery into Mammalian Cells Federica Simeoni, May C. Morris, Frederic Heitz, and Gilles Divita Lentiviral Vector Delivery of siRNA and shRNA Encoding Genes into Cultured and Primary Hematopoietic Cells Mingjie Li and John J. Rossi Exon-Specific RNA Interference: A Tool to Determine the Functional Relevance of Proteins Encoded by Alternatively Spliced mRNAs Alicia M. Celotto, Joo-WonLee, and Brenton R. Graveley Immunoprecipitation of MicroRNPs and Directional Cloning of MicroRNAs Elisavet Maniataki, Maria Dels Angels De Planell Saguer, and Zissimos Mourelatos 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 High-Throughput Analysis of MicroRNA Gene Expression Using Sensitive Probes Mouldy Sioud and Øystein Røsok 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 Index

Blurb

The past decade has witnessed a true revolution in our understanding of how RNA molecules can reduce or silence gene expression, a process that holds great promise in basic research and for antiviral and anticancer applications. In RNA Silencing: Methods and Protocols, many of the most notable and distinguished researchers in the field describe in step-by-step detail their best methods for the design, preparation, and use of RNAs to reduce gene expression in cells and organisms. The techniques range widely and include methods addressing the biochemical aspects of the silencing machinery, RNA silencing in non-mammalian organisms, and the in vivo delivery of siRNAs and silencing vectors. There are also techniques for designing, preparing, and using RNAs to silence gene expression, for fine-tuning regulation by targeting specific isoforms of a given gene, and for the study and use of microRNAs. The protocols follow the successful Methods in Molecular Biology™ series format, each offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Comprehensive and state-of-the-art, RNA Silencing: Methods and Protocols offers today's laboratory investigators a host of optimized and validated techniques to study RNA silencing and translate its concepts into successful practical applications.