Analysis of melanosomal miRNAs

miRNA microarray profiling was performed for MNT-1 cells, pre-mature and mature melanosomes extracted from MNT-1 cells. In order to reveal the miRNAs that are most abundant in mature melanosomes, the most highly expressed miRNAs from mature melanosomes were compared with expression levels in pre-mature melanosomes. The analysis revealed five miRNAs that were highly expressed in mature melanosomes and had been previously associated with melanoma. Total RNA was extracted from MNT-1 cells (2 replicates), pre-mature and mature melanosomes. The melanosomes were either treated with RNase prior to RNA extraction (2 replicates each) or untreated (1 replicate each). Melanosomes were extracted from MNT-1 melanoma cells by sucrose-based density gradient ultracentrifugation. The treatment of melanosomes with RNase prior to RNA extraction was performed in order to remove any kind of RNA that may interact with or stick unspecifically to the outer side of the melanosome membrane. After 10 mins of RNAse treatment, RNase inhibitor was added. The samples of MNT-1 cells and untreated melanosomes were considered for the normalization, but not for further analyses. Microarray Unit of the Core Facility Genomics and Proteomics DKFZ