Quantification of microRNA-21 and microRNA-125b in melanoma tissue
Although microRNAs (miRNAs) have emerged as potent mediators of melanoma development and progression, a precise understanding of their oncogenic role remains unclear. In this study, we analysed formalin-fixed and paraffin-embedded tissues from two separate melanoma cohorts and from a series of benign melanocytic nevi. Using three different quantification methods [array analysis, quantitative PCR (qPCR) and in-situ hybridization (ISH) quantified by digital image analysis], we found considerable miRNA dysregulation in tumours. Using array analysis, samples mainly clustered according to their biological group (benign vs. malignant) and 77 miRNAs differed significantly between nevi and melanoma samples. Increase of miR-21 and miR-142, and decrease of miR-125b, miR-211, miR-101 and miR-513c in the melanomas were verified in both cohorts using qPCR, whereas the decrease of miR-205 observed with array analysis could not be confirmed using qPCR. ISH with digital quantification showed expression of miR-21 and miR-125b in the melanocytic lesions. miR-21 ISH was increased in melanomas, whereas quantification of miR-125b showed uniform ISH expression across nevi and melanomas. Our results support the important involvement of different miRNAs in melanoma biology and may serve as solid basics for further miRNA investigations in melanoma formalin-fixed and paraffin-embedded tissue. In particular, there is increased expression of miR-21 in melanomas compared with benign nevi.