Department of Biomedicine, Aarhus University.
Departments of Oncology.
Melanoma Res. 2020 Feb;30(1):26-38. doi: 10.1097/CMR.0000000000000644.
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.
目前迫切需要新型的诊断性黑素瘤生物标志物，以便在早期预测转移风险增加的可能性。相对定量基因表达是定量验证潜在生物标志物的首选方法。然而，这种方法依赖于稳健的组织特异性参考基因。在黑素瘤领域，由于缺乏经过验证的参考基因，这一直是一个障碍。因此，我们旨在确定用于黑素瘤基因表达归一化的稳健参考基因。我们使用定量实时 PCR 评估了 24 个候选参考基因在 80 个不同厚度、有无溃疡、有无报道转移病例和不同 BRAF 突变状态的福尔马林固定石蜡包埋黑素瘤中的稳健性。我们还评估了多个黑素瘤细胞系中相同基因的表达及其作为归一化剂的稳健性。我们表明，管家基因如 GAPDH 不适合作为黑素瘤中基因表达的独立归一化剂。相反，我们首次确定了一组用于黑素瘤肿瘤和培养的黑素瘤细胞中基因表达归一化的稳健参考基因。我们建议在黑素瘤中使用 CLTA、MRPL19 和 ACTB 的表达几何平均值进行基因表达归一化，在黑素瘤细胞系中使用 CASC3 和 RPS2 的表达几何平均值进行基因表达归一化。根据我们的建议进行归一化将允许通过定量实时 PCR 对潜在的新型黑素瘤生物标志物进行定量验证。
