RNA interference microarrays: high-throughput loss-of-function genetics in mammalian cells.
RNA interference (RNAi) is a biological process in which a double-stranded RNA directs the silencing of target genes in a sequence-specific manner. Exogenously delivered or endogenously encoded double-stranded RNAs can enter the RNAi pathway and guide the suppression of transgenes and cellular genes. This technique has emerged as a powerful tool for reverse genetic studies aimed toward the elucidation of gene function in numerous biological models. Two approaches, the use of small interfering RNAs and short hairpin RNAs (shRNAs), have been developed to permit the application of RNAi technology in mammalian cells. Here we describe the use of a shRNA-based live-cell microarray that allows simple, low-cost, high-throughput screening of phenotypes caused by the silencing of specific endogenous genes. This approach is a variation of "reverse transfection" in which mammalian cells are cultured on a microarray slide spotted with different shRNAs in a transfection carrier. Individual cell clusters become transfected with a defined shRNA that directs the inhibition of a particular gene of interest, potentially producing a specific phenotype. We have validated this approach by targeting genes involved in cytokinesis and proteasome-mediated proteolysis.
Silva
Jose M
JM
Watson School of Biological Sciences, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
Mizuno
Hana
H
Brady
Amy
A
Lucito
Robert
R
Hannon
Gregory J
GJ
D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D046988:Proteasome Endopeptidase Complex; D034622:RNA Interference
D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex