title: Renal cells exposed to cadmium in vitro and in vivo: normalizing gene expression data.
abstract: Cadmium (Cd) is a toxic metal with a long half-life in biological systems. This half-life is partly as a result of metallothioneins (MTs), metal-binding proteins with a high affinity for Cd. The high retention properties of the kidneys reside in proximal tubular cells that possess transport mechanisms for Cd-MT uptake, ultimately leading to more Cd accumulation. Researchers have studied MT-metal interactions using various techniques including quantitative real-time PCR (qPCR), an efficient tool for quantifying gene expression. Often a poor choice of reference genes, which is represented by their instability and condition dependency, leads to inefficient normalization of gene expression data and misinterpretations. This study demonstrates the importance of an efficient normalization strategy in toxicological research. A selection of stable reference genes was proposed in order to acquire reliable and reproducible gene quantification under metal stress using MT expression as an example. Moreover, in vitro and in vivo setups were compared to identify the influence of toxicological compounds in function of the experimental design. This study shows that glyceraldehyde-3-phosphate dehydrogenase (Gapdh), tyrosine monooxygenase/tryptophan5-monooxygenase activation-protein, zeta polypeptide (Ywhaz) and beta-actin (Actb) are the most stable reference genes in a kidney proximal tubular cell line exposed to moderate and high Cd concentrations, applied as CdCl2 . A slightly different sequence in reference gene stability was found in renal cells isolated from rats in vivo exposed to Cd. It was further shown that three reference genes are required for efficient normalization in this experimental setup. This study demonstrates the importance of an efficient normalization strategy in toxicological research.
authors.affiliation: Centre for Environmental Sciences, Hasselt University, Agoralaan Building D, 3590, Diepenbeek, Belgium.
authors.forename: A R
authors.initials: AR
authors.lastname: Nair
authors.forename: K
authors.initials: K
authors.lastname: Smeets
authors.forename: E
authors.initials: E
authors.lastname: Keunen
authors.forename: W-K
authors.initials: WK
authors.lastname: Lee
authors.forename: F
authors.initials: F
authors.lastname: Thévenod
authors.forename: E
authors.initials: E
authors.lastname: Van Kerkhove
authors.forename: A
authors.initials: A
authors.lastname: Cuypers
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002104:Cadmium / Q000633:toxicity*; D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000166:cytology*; D008297:Male; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism; D011916:Rats, Inbred F344; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; C570704:CXXC1 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D015534:Trans-Activators; D014404:Tubulin; C103265:aryl hydrocarbon receptor-interacting protein; D002104:Cadmium; D014446:Tyrosine 3-Monooxygenase; D007041:Hypoxanthine Phosphoribosyltransferase; D019696:Peptidylprolyl Isomerase
keywords: cadmium; metallothionein; qPCR; reference genes