bioRxiv, 2018, published online 14 November
Exposure to graphene oxide sheets alters the expression of reference genes used for real-time RT-qPCR normalization
Background: studies that unravel the interactions between thin, 2D graphene oxide (GO) sheets and the biological milieu, including cells and tissues, are multiplying quickly as the biomedical applications of those and other 2D materials continue to be explored. Many of such studies rely on real-time RT-qPCR as a powerful, yet relatively simple technique to determine gene expression. However, a systematic investigation of potential GO-induced changes in the expression of reference genes, crucial for appropriate normalization of qPCR data that ensures reliability of the results, is still lacking. In this study, we aimed to cover this gap by investigating the stability of the expression of ten (10) candidate reference genes upon exposure to increasing, but subtoxic, concentrations of GO, with two established algorithms (Bestkeeper and NormFinder). The study was performed in a human cancer cell line (MCF7) and in mouse, non-cancerous primary cells (mouse embryonic fibroblasts, MEFs), to assess different behaviors between cell types. Results: Bestkeeper and NormFinder algorithms evidenced significant deviations in the expression of various reference genes. Ribosomal proteins scored among the most significantly dysregulated targets in both cell types. Expression of ACTB and GAPDH, the most frequent calibrators in real-time RT-qPCR studies, was also affected, although differences existed between cell lines. Conclusions: this study illustrates the need to validate reference genes for appropriate real-time RT-qPCR normalization, according to specific experimental conditions, when GO-cell interactions occur.