RNA Interference : Methods for Plants and

The Principles & Protocols Seriesis designed for upper level undergraduates, postgraduates and researchers, particularly those new to a field. Books in the series outline the theoretical background to experimental approaches, followed by bullet-point protocols, which are generic and can be adapted for particular systems. RNA interference (RNAi) has been one of the most exciting and significant new methodologies to appear in the past decade, and it now finds widespread application. This methods manual provides an introduction to the phenomenon to RNA Interference and specific protocols for RNAi, in organisms from plants and C.elegans to Drosophila and mammals. Also included are chapters covering small hairpin RNAs and viral-induced gene silencing.

Tim Doran has worked for his entire career in the field of animal health. He completed his PhD in 1993 at CSIRO Division of Animal Health working on the eradication of bovine tuberculosis in Australia. He then took on a Post-Doctoral Fellowship at St. George's Hospital Medical School in London, where he was awarded the Richard Merkal Fellowship for his outstanding research on paratuberculosis and Johne's disease in cattle.
In 1996 Tim returned to CSIRO Livestock Industries and was the inaugural winner of the 2001 AFFA Science Award for Young People. He is currently project leader of RNAi Technologies at the Australian Animal Health Laboratory. His research team is applying RNAi gene silencing technology to control viral infection in a range of livestock species.

Contributors	p. ix
Introduction	p. xi
RNA interference in plants
Hairpin RNAi in plants	p. 1
Introduction	p. 1
Discovery of hairpin RNAi	p. 1
The molecular mechanisms of hairpin RNAi	p. 2
Using hairpin RNAi in plants	p. 5
Flexible hairpin RNAi methods	p. 9
Applications of hairpin RNAi in plants	p. 12
Summary	p. 15
Virus-induced gene silencing (VIGS) to study gene function in plants	p. 26
Introduction	p. 26
Principles	p. 30
The use of artificial microRNAs in targeted gene silencing	p. 50
Introduction	p. 50
Relevant issues in miRNA biogenesis for amiRNA efficiency	p. 52
The pros and cons of using amiRNAs in gene silencing	p. 62
Future directions to extend amiRNA technology	p. 64
Conlusions	p. 70
RNA interference in animals
RNAi in Caenorhabditis elegans	p. 85
Introduction	p. 85
Delivery of exogenous dsRNA	p. 86
General considerations for the design of an RNAi experiment	p. 88
Large-scale RNAi preparations for biochemical, proteomic and microarray analyses	p. 91
Inducible and tissue-specific RNAi using hairpin transgenes	p. 91
High-throughput RNAi screens in 96-well format	p. 92
RNAi in Drosophila	p. 114
Introduction	p. 114
RNAi in fish and crustaceans	p. 150
Introduction	p. 150
RNAi in crustaceans	p. 150
RNAi in fish	p. 158
Conclusions	p. 166
RNAi in chicken embryos	p. 183
Introduction	p. 183
RNAi in mammalian cells	p. 205
Introduction	p. 205
RNAi and related mechanisms	p. 205
siRNAs and miRNAs: analogous guide RNAs for RNA silencing	p. 206
RNAi in mammalian cells: searching for the right siRNAs	p. 208
RNAi in mammalian cells: getting siRNAs into cells	p. 211
RNAi in mammalian cells: verifying the specificity of RNAi experiments	p. 214
Concluding remarks	p. 214
siRNA and shRNA design	p. 228
Introduction	p. 228
RNAi design methods	p. 230
Index	p. 255
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RNA Interference : Methods for Plants and Animals

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