Melanogenesis Gene Expression Analysis

TRIzol (Invitrogen) was used to obtain the total RNA extract according to the manufacturer’s protocol. To synthesize cDNA, total RNA (2 µg) was reverse-transcribed using oligo dT18 primer. The synthesized cDNA was amplified using a high-capacity cDNA synthesis kit (Bioneer, Daejeon, Korea) with a thermal cycler (Bio-Rad, Hercules, CA, USA). PCR-amplified products were separated using 2% agarose gel, including ethidium bromide (Sigma, St. Louis, MO, USA), and imaged in a gel documentation system (Fujifilm, Tokyo, Japan) [30 (link)]. The following primer sequences were used: mouse tyrosinase 5′-ATAACAGCTCCCACCAGTGC-3′ (sense) and 5′-CCCAGAAGCCAATGCACCTA-3′ (antisense) (NM_011661.5); mouse MITF, 5′-CTGTACTCTGAGCAGCAGGTG-3′ (sense) and 5′-CCCGTCTCTGGAAACTTGATCG-3′ (antisense) (NM_001178049.1); mouse TRP-1 5′-AGACGCTGCACTGCTGGTC AAGCCTGTAGCCCACGTCGTA-3′ (sense) and 5′-GCTGCAGGAGCCTTCTTTCT-3′ (antisense) (NM_001282015.1). The expression of glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous control for qRT-PCR experiments [20 (link)].