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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(TM) 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.
List of contents
Identification of Components of RNAi Pathways Using the Tandem Affinity Purification Method.- Separation of Drosophila RNA Silencing Complexes by Native Gel Electrophoresis.- Preparation and Analysis of Drosha.- RNA Interference in Caenorhabditis elegans.- Down-Regulating Gene Expression by RNA Interference in Trypanosoma brucei.- Analysis of Double-Stranded RNA and Small RNAs Involved in RNA-Mediated Transcriptional Gene Silencing.- Strategies for the Design of Random siRNA Libraries and the Selection of anti-GFP siRNAs.- Silencing Gene Expression with Dicer-Generated siRNA Pools.- Inhibition of Gene Expression In Vivo Using Multiplex siRNA.- Gene Silencing by a DNA Vector-Based RNAi Technology.- Plasmid-Based RNA Interference.- siRNA Delivery In Vivo.- Peptide-Based Strategy for siRNA Delivery into Mammalian Cells.- Lentiviral Vector Delivery of siRNA and shRNA Encoding Genes into Cultured and Primary Hematopoietic Cells.- Exon-Specific RNA Interference.- Immunoprecipitation of MicroRNPs and Directional Cloning of MicroRNAs.- Detection of MicroRNAs and Assays to Monitor MicroRNA Activities In Vivo and In Vitro.- High-Throughput Analysis of MicroRNA Gene Expression Using Sensitive Probes.- Modification of Human U1 snRNA for Inhibition of Gene Expression at the Level of Pre-mRNA.
Summary
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.
Additional text
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)
Report
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)
