Lentiviral shRNA library screen to identify growth-related genes in malignant melanoma

Little is known about genes that promote melanoma cell growth and proliferation. siRNAs may be used to address the role of individual genesin these processes. RNAi library screens were used in the past to gain a comprehensive overview of all genes involved in cell growth, proliferation, migration and other cellular processes. A large-scale loss-of-function screen for eight different melanoma cell lines was performed using a pooled lentiviral shRNA library (GeneNet Human 50K lentiviral shRNA Library,cat#SI206B-1, System Biosciences) to identify genes relevant for melanoma cell growth and proliferation. shRNAs that lead to cell death or reduced growth of transduced melanoma cells are negatively selected and thereby underrepresented in the final cellular shRNA pool and vice versa. The shRNAs of the shRNA library (3-5 per gene) have complementary sequences to probes on the custom Affymetrix microarray HG-U133Plus2 and were analysed using this array. Well-known melanoma cell lines SK-Mel-103, A375, SK-Mel-147, SK-Mel-19, SK-Mel-28, SK-Mel-29, SK-Mel-5, WM3523cln6 were transduced with the lentiviral shRNA library and grown for 10 days under puromycin selection (day 10), control cells of respective cell lines were transduced and frozen immediately after transduction and genomic integration of shRNAs (day 0). Totel DNA was extracted and genomically integrated shRNAs were hybridized to Affymetrix microarrays (HG-U133Plus2.0 array).