Rna Silencing

Maximizing the potential of RNA interference in functional genomics - as well as in the development of therapeutics - continues to be at the forefront of biomedical research. Unlike journal articles, Gene Silencing by RNA Interference: Technology and Application combines essential background to the RNAi field with practical techniques designed by r

In the past three years, the use of double-stranded RNA to silence gene activity has become widely and rapidly adopted. RNA interference is highly specific and remarkably potent, and it acts on cells and tissues far removed from the site of introduction. The principles behind RNAi are just being uncovered, but this laboratory technique has been applied effectively in a wide variety of animal and plant species. Variations on RNAi are revolutionizing many approaches to experimental biology, complementing traditional genetic technologies with a quicker and less expensive way of mimicking the effects of mutations both in cell cultures and in living animals. Recent advances in the use of RNAi to engineer heritable silencing in mammals, to alter stem cells for organ reconstitution, and to alter the course of disease in model systems indicate that RNAi may have a future in disease therapy. Written by pioneers in this new field and edited by Gregory Hannon, one of its leading figures, RNAi: A Guide to Gene Silencing presents the principles of RNAi and reliable protocols for its laboratory use in Caenorhabditis elegans, Drosophila, plants, avian embryos, mammalian cells, mouse oocytes, and more. This important and unique book is an essential laboratory resource for scientists studying gene regulation and for all experimental biologists interested in the emerging practical applications of RNAi.

This book focuses on an emerging, central issue in molecular genetics and the development of eukaryotes: the control of gene expression by small species of RNA. As an exciting new field of endeavor, it is the first book by a single author to deal comprehensively with RNA silencing. The book provides the historical background of the field preceding the seminal work by Fire and associates in 1998 on the impact of small double-stranded RNA on the expression of nematode genes, which is considered the beginning of RNA silencing research. RNA silencing is described in a wide range of plants and animals including protozoa, simple metazoa, insects, non-mammalian vertebrates, and mammals. In each case the experimental results are provided with the accompanying background and with illustrations. There is also an appendix on the prospective use of RNA silencing in gene therapy, which is intended as a guide for investigators wishing to explore this possibility.

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 BiologyTM 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.

RNAi technology is used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division. Exploitation of the pathway is also a promising tool in biotechnology and medicine. Introducing new technology in the study of RNA

This methods manual provides an introduction to RNA interference, the theory behind its many applications, and specific protocols for RNAi, in organisms from plants and C.elegans to Drosophila and mammals. There are also chapters covering small hairpin RNAs and viral-induced gene silencing.

In the last few years the major effect that RNAi has had in invertebrate systems is beginning to take hold in mammalian systems through both single gene knockdown experiments and genome-scale screens. In the next decade, there will no doubt be both notable successes and failures as we attempt to apply this genetic tool to various biological problems. Through the introduction of RNAi, mammalian systems have finally gained admittance to the pantheon of model genetic systems.

This 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 examples of successful experiments and troubleshooting hints to help in establishing your own successful RNAi experiments. Also includes a list of suppliers for RNAi reagents and equipment as well as a glossary of terms.