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Cytoglobin (CYGB) is a ubiquitously expressed protein with a protective role against oxidative stress, fibrosis and tumor growth, shown to be transcriptionally regulated under hypoxic conditions. Hypoxia-inducible  CYGB  expression is observed in several cancer cell lines and particularly in various melanoma-derived cell lines. However, reliable detection of hypoxia-inducible mRNA levels by qPCR depends on the critical choice of suitable reference genes for accurate normalization. Limited evidence exists to support selection of the commonly used reference genes in hypoxic models of melanoma. This study aimed to select the optimal reference genes to study  CYGB  expression levels in melanoma cell lines exposed to hypoxic conditions (0.2% O 2 ) and to the HIF prolyl hydroxylase inhibitor roxadustat (FG-4592). The expression levels of candidate genes were assessed by qPCR and the stability of genes was evaluated using the geNorm and NormFinder algorithms. Our results display that  B2M  and  YWHAZ  represent the most optimal reference genes to reliably quantify hypoxia-inducible  CYGB  expression in melanoma cell lines. We further validate hypoxia-inducible CYGB expression on protein level and by using  CYGB  promoter-driven luciferase reporter assays in melanoma cell lines.

A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma