MiR-16 is not a suitable reference for normalization of serum microRNA (miRNA) expression in melanoma patients. This study evaluated miR-16 as a potential reference gene for normalization of serum miRNA in melanoma patients. A total of 143 primary cutaneous melanoma patients and 60 control subjects were studied. The control group included healthy volunteers, rheumatoid arthritis patients, non-melanoma cancer patients, and Atypical Mole Syndrome patients. miR-16 expression was analyzed using qRT-PCR. The Kruskal-Wallis test and Wilcoxon test with Bonferroni correction were used to compare miR-16 expression between melanoma patients and control groups. The results showed no significant difference in miR-16 expression between melanoma patients and healthy volunteers (p = 0.37). However, miR-16 expression was significantly different across melanoma stages (p = 0.015). An equivalence test was performed to determine if miR-16 expression was equivalent between melanoma patients and control groups, but no equivalence was found. The study concludes that miR-16 cannot be used as a universal normalizer in serum-based miRNA studies of melanoma patients. The study highlights the importance of selecting appropriate reference genes for normalization in miRNA studies, as the stability of miRNAs in serum can be influenced by various factors. The study also notes that RNU6B, a commonly used reference gene in tissue-based studies, is not consistently detected in plasma or serum. The findings suggest that further research is needed to identify suitable reference genes for normalization in miRNA-based melanoma studies.MiR-16 is not a suitable reference for normalization of serum microRNA (miRNA) expression in melanoma patients. This study evaluated miR-16 as a potential reference gene for normalization of serum miRNA in melanoma patients. A total of 143 primary cutaneous melanoma patients and 60 control subjects were studied. The control group included healthy volunteers, rheumatoid arthritis patients, non-melanoma cancer patients, and Atypical Mole Syndrome patients. miR-16 expression was analyzed using qRT-PCR. The Kruskal-Wallis test and Wilcoxon test with Bonferroni correction were used to compare miR-16 expression between melanoma patients and control groups. The results showed no significant difference in miR-16 expression between melanoma patients and healthy volunteers (p = 0.37). However, miR-16 expression was significantly different across melanoma stages (p = 0.015). An equivalence test was performed to determine if miR-16 expression was equivalent between melanoma patients and control groups, but no equivalence was found. The study concludes that miR-16 cannot be used as a universal normalizer in serum-based miRNA studies of melanoma patients. The study highlights the importance of selecting appropriate reference genes for normalization in miRNA studies, as the stability of miRNAs in serum can be influenced by various factors. The study also notes that RNU6B, a commonly used reference gene in tissue-based studies, is not consistently detected in plasma or serum. The findings suggest that further research is needed to identify suitable reference genes for normalization in miRNA-based melanoma studies.