4.6. Quantitative Polymerase Chain Reaction (qPCR) Analysis

Total RNA extracts were prepared from the harvested cells using a column-based isolation kit (RNeasy Mini Kit; Qiagen, Hilden, Germany) according to the manufacturer’s instructions [32]. After quantification at 260 nm wavelength, RNA (0.5 μg) was reverse-transcribed to cDNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Thermo Fisher Scientific) with oligo(dT)_12–18 primer. To analyze the relative mRNA levels for each gene, SYBR Green-based real-time PCR was performed using LightCycler^® Multiplex Masters (Roche, Basel, Switzerland) with the designated primer sets (Table 1) on LightCycler^® Nano Instrument (Roche). The transcript expression levels were normalized with the expression level of β-actin.

Primer sets for real-time PCR.

Tyr, tyrosinase; Tyrp1, tyrosinase-related protein 1; Tyrp2, tyrosinase-related protein 1; Mitf, microphthalmia-associated transcription factor.