Silva, J. M., Mizuno, H., Brady, A., Lucito, R., Hannon, G. J. (April 2004) RNA interference microarrays: high-throughput loss-of-function genetics in mammalian cells. Proc Natl Acad Sci U S A, 101 (17). pp. 6548-52. ISSN 0027-8424 (Print)
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Abstract
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.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Animals Cell Cycle Cell Line Cysteine Endopeptidases metabolism Genes, Reporter Humans Multienzyme Complexes metabolism Oligonucleotide Array Sequence Analysis Proteasome Endopeptidase Complex RNA Interference |
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNAi bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > dsRNA bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene silencing |
| CSHL Authors: | |
| Communities: | CSHL labs > Hannon lab |
| Depositing User: | CSHL Librarian |
| Date: | 27 April 2004 |
| Date Deposited: | 26 Jan 2012 15:32 |
| Last Modified: | 09 Nov 2017 17:12 |
| PMCID: | PMC404082 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/22482 |
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