miRNA profiling of Melanoma Cell lines and Normal Melanocytes

To identify microRNAs potentially involved in melanomagenesis we compared microRNA transcription profiles between melanoma cell lines and cultured melanocytes. miRNA microarrays were manufactured by Agilent Technologies (Santa Clara, CA), and contain 20-40 features targeting each of 470 human miRNAs (Agilent design ID 016436). Sequences of the 470 miRNAs were obtained from the Sanger miRBase, release 9.1. Labeling and hybridization of total RNA samples were performed according to the manufacturer’s protocol. 100ng total RNA was used as input into the labeling reaction, and the entire reaction was hybridized to the array for 20 hours at 55°C. The labeling and hybridizations of the nine human tissues were done 4-5 times using the same starting RNA sample for each tissue. Total RNA was extracted from 51 melanoma cells and 2 pools of melanocytes using the QIAGEN miRNA kits.