RNA Extraction and RT-qPCR Analysis of Murine Melanogenic Genes

Total RNA was extracted using TRIzol reagent (Invitrogen), in accordance with the manufacturer’s instructions. To obtain cDNA, total RNA (2 µg) was reverse-transcribed using an oligo(dT) primer. The cDNA was amplified using the High-Capacity cDNA synthesis kit (Bioneer, Daejeon, Korea) in a PCR machine (Bio-Rad, Hercules, CA, USA). PCR was performed using a PCR premix (Bioneer), and real-time RT-PCR was performed using the StepOne model (Applied Biosystems, Foster City, CA, USA) and SYBR Green premix, according to the manufacturer’s instructions (Applied Biosystems). Primers were synthesized by Bioneer. The following primer sequences were used: mouse tyrosinase: 5′-ATAACAGCTCCCACCAGTGC-3′ (sense) and 5′-CCCAGAAGCCAATGCACCTA-3′ (antisense); mouse MITF: 5′-CTGTACTCTGAGCAGCAGGTG-3′ (sense) and 5′-CCCGTCTCTGGAAACTTGATCG-3′ (antisense); and mouse TRP-1: 5′-AGACGCTGCACTGCTGGTCAAGCCTGTAGCCCACGTCGTA-3′ (sense) and 5′-GCTGCAGGAGCCTTCTTTCT-3′ (antisense). The expression of glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous control for qRT-PCR [30 (link)].