High-throughput RNA interference strategies for target discovery and validation by using synthetic short interfering RNAs: functional genomics investigations of biological pathways.
During the past five years, RNA interference (RNAi) has emerged as arguably the best functional genomics tool available to date, providing direct, causal links between individual genes and loss-of-function phenotypes through robust, broadly applicable, and readily upscalable methodologies. Originally applied experimentally in C. elegans and Drosophila, RNAi is now widely used in mammalian cell systems also. The development of commercially available libraries of short interfering RNAs (siRNAs) and other RNAi silencing reagents targeting entire classes of human genes provide the opportunity to carry out genome-scale screens to discover and characterize gene functions directly in human cells. A key challenge of these studies, also faced by earlier genomics or proteomics approaches, resides in reaching an optimal balance between the necessarily high throughput and the desire to achieve the same level of detailed analysis that is routine in conventional small-scale studies. This chapter discusses technical aspects of how to perform such screens, what parameters to monitor, and which readouts to apply. Examples of homogenous assays and multiplexed high-content microscopy-based screens are demonstrated.
Sachse
Christoph
C
Cenix BioScience GmbH, Dreseden, Germany.
Krausz
Eberhard
E
Krönke
Andrea
A
Hannus
Michael
M
Walsh
Andrew
A
Grabner
Anne
A
Ovcharenko
Dmitriy
D
Dorris
David
D
Trudel
Claude
C
Sönnichsen
Birte
B
Echeverri
Christophe J
CJ
D017209:Apoptosis; D001331:Automation; D002455:Cell Division; D045744:Cell Line, Tumor; D017930:Genes, Reporter; D023281:Genomics; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
D034741:RNA, Small Interfering