Sensimix sybr reagent

The level of CYBB mRNA was quantified using two different primer pairs, compared with the level of the endogenous control, β-Actin (Eurogentec), and expressed as relative quantities compared with the WT cell line using the ΔΔCT method [21] (link). Primers used included gp91 exon 1 forward (CAACACATTCAACCTCTGCC), gp91 exon 3 reverse (GGACAGCAGATTTCGACAG), gp91 exon1-2 boundary forward (TTTTGTCATTCTGGTTTGGCTG), and gp91 exon 2-3 boundary reverse (CCAGTGCTGACCCAAGAAGT). Reactions were carried out in triplicate on an ABI StepOne Plus qPCR machine using SensiMix SYBR reagent (Bioline).

RNA was isolated using TRIZOL^® (Invitrogen, Carlsbad, CA, USA) and quantitative real-time PCR performed using SENSIMIX SYBR reagent (Bioline, Boston, MA, USA). Expression levels were quantified relative to glyceraldehyde 3-phosphate dehydrogenase via the Livak method. The following primer sequences were used: MITF, 5′-CCGTCTCTCACTGGATTGGT-3′, 5′-TACTTGGTGGGGTTTTCGAG-3′; TRPM1, 5′-CACCCAGAGCTACCCAACAGA-3′, 5′-CGGATATACATGGCTTTATTGG-3′; PMEL, 5′-CTGGATGGTACAGCCACCTT-3′, 5′-GGCACTTTCAATACCCTGGA-3′; TYR, 5′-CTGGAAGGATTTGCTAGTCCAC-3′, 5′-CCTGTACCTGGGACATTGTTC-3′; MLANA, 5′-TCTGGGCTGAGCATTGGG-3′, 5′-AGACAGTCACTTCCATGGTGTGTG-3′; CDK2, 5′-ATGGAGAACTTCCAAAAGGTGGA-3′, 5′-CAGGCGGATTTTCTTAAGCG-3′; CCND1, 5′-GAACTACCTGGACCGCTTCCT-3′, 5′-TTCGATCTGCTCCTGGCAGG-3′; BCL2A1, 5′-CGTAGACACTGCCAGAACACTA-3′, 5′-GGGCAATTTGCTGTCGTAGA-3′; CTGF, 5′-CGACTGGAAGACACGTTTGG-3′, 5′-ATCCCACAGGTCTTGGAACA-3′; NEGR, 5′-GGTCAGTGGATCCTCGAGTT-3′, 5′-TGGGCCATCATCTGTCACAT-3′; CYR61, 5′-TGCGAAGATGGGGAGACATT-3′, 5′-CTGTAGAAGGGAAACGCTGC-3′; CTGF promoter, 5′-GCCAATGAGCTGAATGGAGT-3′, 5′-CAATCCGGTGTGAGTTGATG-3′; CYR61 promoter, 5′-AGCAAACAGCTCACTGCCTT-3′, 5′-ATGGTAGTTGGAGGGTCGTG-3′; MITF promoter, 5′-GCAGTTATTCGGCCATTGGA-3′, 5′-GGAAGCCCTACGAGTTTGGT-3′.