Real-time PCR (RT-PCR)
Total RNA was extracted from cochleae using TRIzol reagent (Invitrogen,
USA). Random hexamers were used for synthesizing the first cDNA strand
synthesis with 1 μg of total RNA (Molecular Biology). Then, the
expression of an array of genes (MITF , SOX10 , PAX3 ,TYR , DCT , and MC1R ) involved in the MITF signaling
pathway was evaluated using EvaGreen Supermix on a CFX96 apparatus
(Bio-Rad). The thermal cycling profile was 95 °C for 30 s, followed by
40 cycles of 95 °C for 5 s, 58 °C for 10 s, and extension at 95 °C for 5
s, 65 °C to 95 °C, and 0.5 °C for 5 s. The PCR primers are listed in
Table S1. The expression of each target gene in WT and mutant
littermates was standardized against that of actin mRNA using the
2-ΔΔCt method.