title: Identification of reference genes for quantitative real-time PCR studies in human cell lines under copper and zinc exposure.
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.
authors: Laboratorio de Bioinformática y Expresión Génica, INTA, Universidad de Chile, Santiago, Chile. tdelpozo@inta.uchile.cl.
authors: Talía
authors: T
authors: Del Pozo
authors: Laboratorio de Bioinformática y Expresión Génica, INTA, Universidad de Chile, Santiago, Chile. rgutier2@smail.uni-koeln.de.|Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany. rgutier2@smail.uni-koeln.de.
authors: Ricardo
authors: R
authors: Gutiérrez-Garcia
authors: Laboratorio de Bioinformática y Expresión Génica, INTA, Universidad de Chile, Santiago, Chile.|Center for Genome Regulation, Universidad de Chile, Santiago, Chile.|Mathomics, Center for Mathematical Modeling, Universidad de Chile, Santiago, Chile.
authors: Mauricio
authors: M
authors: Latorre
authors: Laboratorio de Bioinformática y Expresión Génica, INTA, Universidad de Chile, Santiago, Chile.|Center for Genome Regulation, Universidad de Chile, Santiago, Chile.
authors: Mauricio
authors: M
authors: González
authors: Nutrition and Dietetics, Faculty of Health Sciences, San Sebastian University, Valdivia, Chile.
authors: Miriam
authors: M
authors: Suazo
mesh_terms: D002460:Cell Line; D003300:Copper / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism / Q000592:standards; D020869:Gene Expression Profiling; D006801:Humans; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism / Q000592:standards; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015032:Zinc / Q000494:pharmacology*
chemical_list: C506526:ATP5F1B protein, human; D003574:Cytochrome c Group; D003300:Copper; D025261:Mitochondrial Proton-Translocating ATPases; D015032:Zinc
keywords: Gene expression; Gene reference; Human cell lines; Metal exposure; Real-time RT-PCR