Abstract
Accurate quantification depends on normalization of the measured gene expression data. In particular, gene expression studies with exposure to metals are challenging due their toxicity and redox-active properties. Here, we assessed the stability of potential reference genes in three cell lines commonly used to study metal cell metabolism: Caco-2 (colon), HepG2 (liver) and THP-1 (peripheral blood) under copper (Cu) or zinc (Zn) exposure. We used combined statistical tools to identify the best reference genes from a set of eleven candidates, which included traditional “housekeeping” genes such as GAPDH and B-ACTIN, in cell lines exposed to high and low, Zn and Cu concentrations. The expression stabilities of ATP5B (ATP synthase) and CYC1 (subunits of the cytochrome) were the highest considering the effect of Zn and Cu treatments whereas SDHA (succinate dehydrogenase) was found to be the most unstable gene. Even though the transcriptional effect of Zn and Cu is very different in term of redox properties, the same best reference genes were identified when Zn or Cu treatments were analyzed together. Our results indicate that ATP5B/CYC1 are the best candidates for reference genes after metal exposure, which can be used as a suitable starting point to evaluate gene expression with other metals or in different cell types in human models.
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Abbreviations
- Cu:
-
Copper
- Cu-His:
-
Copper-histidine complex
- Zn:
-
Zinc
- qPCR:
-
Quantitative real-time PCR
- HKG:
-
Housekeeping genes
- Ct:
-
Threshold cycle
- CV:
-
Coefficient of variation
- ROS:
-
Reactive oxygen species
- ACTB :
-
β-actin
- GAPDH :
-
Glyceraldehyde 3-phosphate dehydrogenase
- B2M :
-
β-2- microglobulin
- CYC1 :
-
Cytochrome c1
- ATP5B :
-
β-ATP synthase subunit
- YWHAZ :
-
Phospholipase A2
- HPRT1 :
-
Hypoxanthine-phosphoribosyltransferase 1
- TOP1 :
-
Topoisomerase (DNA) I
- HMBS :
-
Hydroxymethylbilane synthase
- SDHA :
-
Succinate dehydrogenase complex, subunit A,
- UBC :
-
Ubiquitin C
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Acknowledgments
This work was supported by Chilean Grant Fondecyt 11070255, 3120098, 11150679 and FONDAP 15090007. Authors thank Ms. Estela Blanco for editing.
Authors’ contributions
TDP, ML, MG and MS conducted the research and analyzed the data; RG performed experiments and analyzed the data; TDP and ML wrote the paper; TDP was responsible for manuscript preparation. All authors read and approved the final version.
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Talía del Pozo and Ricardo Gutiérrez-Garcia have contributed equally to this paper.
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del Pozo, T., Gutiérrez-Garcia, R., Latorre, M. et al. Identification of reference genes for quantitative real-time PCR studies in human cell lines under copper and zinc exposure. Biometals 29, 935–944 (2016). https://doi.org/10.1007/s10534-016-9965-x
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DOI: https://doi.org/10.1007/s10534-016-9965-x