Comparison of variability and sensitivity between nuclear translocation and luciferase reporter gene assays.
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Unterreiner, Vincent, Ibig-Rehm, Yvonne, Simonen, Marjo, Gubler, Hanspeter and Gabriel, Daniela (2009) Comparison of variability and sensitivity between nuclear translocation and luciferase reporter gene assays. Journal of Biomolecular Screening : the official journal of the Society for Biomolecular Screening, 14 (1). pp. 59-65. ISSN 1087-0571
Abstract
High-content screening (HCS), a technology based on subcellular imaging by automated microscopy and sophisticated image analysis, has emerged as an important platform in small-molecule screening for early drug discovery. To validate a subcellular imaging assay for primary screening campaigns, an HCS assay was compared with a non-image-based readout in terms of variability and sensitivity. A study was performed monitoring the accumulation of the forkhead transcription factor of the O subfamily (FOXO3a) coupled with green fluorescent protein in the nucleus of human osteosarcoma (U-2 OS) cells. In addition, the transcription of a luciferase gene coupled with a FOXO3a-responsive promoter was monitored. This report demonstrates that both assay formats show good reproducibility in primary and concentration response screening despite differences in statistical assay quality. In primary screening, the correlation of compound activity between the 2 assays was low, in contrast to the good correlation of the IC(50) values of confirmed compounds. Furthermore, the high-content imaging assay showed a mean shift of 2.63-fold in IC(50) values compared with the reporter gene assay. No chemical scaffold was specifically found with 1 of the technologies only, however these results validate the HCS technology against established assays for screening of new molecular entities.
| Item Type: | Article |
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| Additional Information: | author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used |
| Keywords: | high-content screening; reporter gene assay; correlation; agreement; sensitivity |
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| Date Deposited: | 14 Dec 2009 13:48 |
| Last Modified: | 31 Jan 2013 00:53 |
| URI: | https://oak.novartis.com/id/eprint/1511 |