Identification of robust reference genes for studies of gene expression in FFPE melanoma samples and melanoma cell lines

There is an urgent need for novel diagnostic melanoma biomarkers that can predict increased risk of metastasis at an early stage. Relative quantification of gene expression is the preferred method for quantitative validation of potential biomarkers. However, this approach relies on robust tissue-specific reference genes. In the melanoma field, this has been an obstacle due to lack of validated reference genes. Accordingly, we aimed to identify robust reference genes for normalization of gene expression in melanoma. The robustness of 24 candidate reference genes was evaluated across 80 formalin-fixed paraffin-embedded melanomas of different thickness, −/+ ulceration, −/+ reported cases of metastases and of different BRAF mutation status using quantitative real-time PCR. The expression of the same genes and their robustness as normalizers was furthermore evaluated across a number of melanoma cell lines. We show that housekeeping genes like GAPDH do not qualify as stand-alone normalizers of genes expression in melanoma. Instead, we have as the first identified a panel of robust reference genes for normalization of gene expression in melanoma tumors and cultured melanoma cells. We recommend using a geometric mean of the expression of CLTA, MRPL19 and ACTB for normalization of gene expression in melanomas and a geometric mean of the expression of CASC3 and RPS2 for normalization of gene expression in melanoma cell lines. Normalization, according to our recommendation will allow for quantitative validation of potential novel melanoma biomarkers by quantitative real-time PCR.