Use of high-throughput RT-qPCR to assess modulations of gene expression profiles related to genomic stability and interactions by cadmium
- PMID: 26525392
- PMCID: PMC5065590
- DOI: 10.1007/s00204-015-1621-7
Use of high-throughput RT-qPCR to assess modulations of gene expression profiles related to genomic stability and interactions by cadmium
Abstract
Predictive test systems to assess the mode of action of chemical carcinogens are urgently required. Within the present study, we applied the Fluidigm dynamic array on the BioMark™ HD System for quantitative high-throughput RT-qPCR analysis of 95 genes and 96 samples in parallel, selecting genes crucial for maintaining genomic stability, including stress response as well as DNA repair, cell cycle control, apoptosis and mitotic signaling. The specificity of each individually designed sequence-specific primer pair and their respective target amplicons were evaluated via melting curve analysis as part of qPCR and size verification via agarose gel electrophoresis. For each gene, calibration curves displayed high efficiencies and correlation coefficients in the identified linear dynamic range as well as low intra-assay variations. Data were processed via Fluidigm real-time PCR analysis and GenEx software, and results were depicted as relative gene expression according to the ΔΔC q method. Subsequently, gene expression analyses were conducted in cadmium-treated adenocarcinoma A549 and epithelial bronchial BEAS-2B cells. They revealed distinct dose- and time-dependent and also cell-type-specific gene expression patterns, including the induction of genes coding for metallothioneins, the oxidative stress response, cell cycle control, mitotic signaling and apoptosis. Interestingly, while genes coding for the DNA damage response were induced, distinct DNA repair genes were down-regulated at the transcriptional level. Thus, this approach provided a comprehensive overview on the interaction by cadmium with distinct signaling pathways, also reflecting molecular modes of action in cadmium-induced carcinogenicity. Therefore, the test system appears to be a promising tool for toxicological risk assessment.
Keywords: Cadmium; Fluidigm dynamic array; Gene expression profiling; Genomic stability; High-throughput RT-qPCR.
Conflict of interest statement
Compliance with ethical standard Conflict of interest The authors declare that they have no competing interests.
Figures
![Fig. 1](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/5065590/bin/204_2015_1621_Fig1_HTML.gif)
![Fig. 2](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/5065590/bin/204_2015_1621_Fig2_HTML.gif)
![Fig. 3](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/5065590/bin/204_2015_1621_Fig3_HTML.gif)
![Fig. 4](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/5065590/bin/204_2015_1621_Fig4_HTML.gif)
![Fig. 5](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/5065590/bin/204_2015_1621_Fig5_HTML.gif)
![Fig. 6](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/5065590/bin/204_2015_1621_Fig6_HTML.gif)
![Fig. 7](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/5065590/bin/204_2015_1621_Fig7_HTML.gif)
![Fig. 8](https://www.ncbi.nlm.nih.gov/pmc/articles/instance/5065590/bin/204_2015_1621_Fig8_HTML.gif)
Similar articles
-
Activation of AIFM2 enhances apoptosis of human lung cancer cells undergoing toxicological stress.Toxicol Lett. 2016 Sep 6;258:227-236. doi: 10.1016/j.toxlet.2016.07.002. Epub 2016 Jul 5. Toxicol Lett. 2016. PMID: 27392435
-
Cadmium induced cell apoptosis, DNA damage, decreased DNA repair capacity, and genomic instability during malignant transformation of human bronchial epithelial cells.Int J Med Sci. 2013 Aug 30;10(11):1485-96. doi: 10.7150/ijms.6308. eCollection 2013. Int J Med Sci. 2013. Retraction in: Int J Med Sci. 2014 Jan 15;11(3):246. doi: 10.7150/ijms.8533. PMID: 24046522 Free PMC article. Retracted.
-
Molecular Mechanisms of Malignant Transformation by Low Dose Cadmium in Normal Human Bronchial Epithelial Cells.PLoS One. 2016 May 17;11(5):e0155002. doi: 10.1371/journal.pone.0155002. eCollection 2016. PLoS One. 2016. PMID: 27186882 Free PMC article.
-
Mechanisms of cadmium induced genomic instability.Mutat Res. 2012 May 1;733(1-2):69-77. doi: 10.1016/j.mrfmmm.2011.09.002. Epub 2011 Sep 17. Mutat Res. 2012. PMID: 21945723 Review.
-
Cadmium and cancer.Met Ions Life Sci. 2013;11:491-507. doi: 10.1007/978-94-007-5179-8_15. Met Ions Life Sci. 2013. PMID: 23430782 Review.
Cited by
-
Effect of Long-Term Low-Dose Arsenic Exposure on DNA Methylation and Gene Expression in Human Liver Cells.Int J Mol Sci. 2023 Oct 16;24(20):15238. doi: 10.3390/ijms242015238. Int J Mol Sci. 2023. PMID: 37894918 Free PMC article.
-
Arsenite Impairs BRCA1-Dependent DNA Double-Strand Break Repair, a Mechanism Potentially Contributing to Genomic Instability.Int J Mol Sci. 2023 Sep 21;24(18):14395. doi: 10.3390/ijms241814395. Int J Mol Sci. 2023. PMID: 37762697 Free PMC article.
-
Impact of Manganese and Chromate on Specific DNA Double-Strand Break Repair Pathways.Int J Mol Sci. 2023 Jun 20;24(12):10392. doi: 10.3390/ijms241210392. Int J Mol Sci. 2023. PMID: 37373538 Free PMC article.
-
Gene Expression Profiling of Mono- and Co-Culture Models of the Respiratory Tract Exposed to Crystalline Quartz under Submerged and Air-Liquid Interface Conditions.Int J Mol Sci. 2022 Jul 14;23(14):7773. doi: 10.3390/ijms23147773. Int J Mol Sci. 2022. PMID: 35887123 Free PMC article.
-
Impact of Nano- and Micro-Sized Chromium(III) Particles on Cytotoxicity and Gene Expression Profiles Related to Genomic Stability in Human Keratinocytes and Alveolar Epithelial Cells.Nanomaterials (Basel). 2022 Apr 11;12(8):1294. doi: 10.3390/nano12081294. Nanomaterials (Basel). 2022. PMID: 35458002 Free PMC article.
References
-
- Andersen CL, Jensen JL, Orntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004;64:5245–5250. doi: 10.1158/0008-5472.CAN-04-0496. - DOI - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical