RNA Extraction and qPCR Analysis

Total skin tissue RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), as described previously [24 (link),40 (link)]. RNA concentration and quality were analyzed using a CFX96^TM Real-Time System (Bio-Rad, Hercules, CA, USA). The sample was treated with recombinant DNase I (DNA-free; Ambion, Austin, TX, USA) to remove contaminating DNA, and RNA was reverse-transcribed using a reagent High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. A total of 50 PCR cycles were performed as follows: 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 30 s for denaturation, annealing, and extension, respectively. The primers used are listed in Table S1.