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Quantitative analysis of gene expression changes in response to genotoxic compounds.
- Source :
-
Toxicology in vitro : an international journal published in association with BIBRA [Toxicol In Vitro] 2017 Mar; Vol. 39, pp. 15-28. Date of Electronic Publication: 2016 Nov 05. - Publication Year :
- 2017
-
Abstract
- Techniques that quantify molecular endpoints sufficiently sensitive to identify and classify potentially toxic compounds have wide potential for high-throughput in vitro screening. Expression of three genes, RAD51C, TP53 and cystatin A (CSTA), in HEPG2 cells was measured by Q-PCR amplification. In parallel, we developed alternative assays for the same 3 gene signature based on an acridinium-ester chemiluminescent reporter molecule. HEPG2 cells were challenged with eighteen different compounds (n=18) chosen to represent compounds that are genotoxic (n=8), non-genotoxic non-carcinogenic (n=2) or have a less well defined mechanism of action with respect to genotoxicity (n=8). At least one of the three genes displayed dysregulated expression in the majority of compounds tested by Q-PCR and ten compounds changed the CSTA expression significantly. Acridinium-ester labelled probes for the three genes were synthesised and tested. Analytical sensitivity was characterised and suggested a limit of detection generally better than 0.1fmol but often 10-50 attomol. A linear amplification step was optimised and this quantitative method detected statistically significant increases in RAD51C and CSTA expression in agreement with the Q-PCR results, demonstrating the potential of this technology. The broad agreement of the amplified chemiluminescent method and Q-PCR in measuring gene expression suggests wider potential application for this chemiluminescent technology.<br /> (Copyright © 2016 Elsevier B.V. All rights reserved.)
Details
- Language :
- English
- ISSN :
- 1879-3177
- Volume :
- 39
- Database :
- MEDLINE
- Journal :
- Toxicology in vitro : an international journal published in association with BIBRA
- Publication Type :
- Academic Journal
- Accession number :
- 27825931
- Full Text :
- https://doi.org/10.1016/j.tiv.2016.11.004