Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: Is β-actin a reliable normalizer for photoaging studies?

► UVA modulates expression of genes involved in dermal re-modelling. ► Accurate gene expression evaluation requires suitable reference genes. ► Human dermal fibroblasts (HDF) cultured under 21% O 2 and under physiological 5% O 2. ► Most suitable reference genes identified for qPCR analysis on HDF, UVA-treated. ► β-Actin is unsuitable as reference gene for UVA-treated HDF. Data normalization of gene expression on human dermal fibroblasts (HDF) exposed to UVA has commonly been done using either GAPDH or β-actin as reference genes without any validation of their expression stability. Since this aspect, important for accurate normalization, has been overlooked, we aimed to establish a suitable set of reference genes for studies on UVA-treated HDF cultured under both standard atmospheric oxygen tension (normoxia, 21%) and under a physiological, low oxygen tension for these cells (hypoxia, 5%). The stability of six commonly used reference genes was assessed using the geNorm and NormFinder softwares subsequent to reverse-transcription quantitative real-time PCR (RT-qPCR). GAPDH/SDHA were found to be the most stable genes under normoxia, while SDHA/TBP or HPRT1/β2M were the most stable ones under hypoxia in HDF exposed to 18 J/cm 2 UVA. β-Actin was always the most unstable reference gene. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of MMP-1 and COL1A1 were analyzed vs the best reference genes and the worst one. These reference genes are hence recommended for future qPCR analyses in studies concerning photo-damage on UVA-treated HDF.