Analysis of stably expressed genes with low-dose etoposide for toxicological studies in osteosarcoma

Osteosarcoma is an aggressive bone malignancy with largest incidence in children and adolescents. Patients' poor survival rates are mostly due to poor responsiveness to current chemotherapy. For this reason, gene expression studies are necessary to elucidate the mechanisms of chemosensitivity and chemoresistance. However, for gene expression studies to be reliable, it is necessary to normalise all data with stably expressed reference genes. The aim of the study was to identify stably expressed genes suitable for use as normalisers in toxicological studies using osteosarcoma in vitro models. Osteosarcoma MG-63, Saos-2 and U2OS cell lines were exposed to 1µM etoposide for 48h, a dose below IC₂₅. Gene expression was determined by quantitative PCR (qPCR). Four different methods were used to analyse the cycle threshold values obtained by qPCR: the comparative ΔCt, geNorm, NormFinder and BestKeeper. Exposure to etoposide resulted in expression changes in a subgroup of genes. Depending on the conditions used different genes were the most stably expressed across cell lines. Nevertheless, other genes commonly used as reference genes showed higher variability, pointing to the need for screening reference genes.

Acknowledgements: This work was supported by the Portuguese Foundation for Science and Technology (FCT), through the post-doctoral fellowship of J.M.P. Ferreira de Oliveira (SFRH/BPD/74868/2010).