RNA isolation and qPCR analysis

Skin samples collected from the same anatomical locations, including epidermis and dermis, were used and compared in the experiments. After excision, skin tissues were immediately placed in RNAlater solution (Ambion, Austin, TX), stored at −80°C and used later. To extract RNA, skin samples were placed in liquid nitrogen and ground with a mortar and pestle. Total RNA was then isolated with Trizol reagent (Invitrogen, Carlsbad, CA) and further purified according to the manufacturer’s instructions. Reverse transcription was performed using 2 μg total RNA for first strand cDNA synthesis with M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) in a total volume of 25 μL. A portion of the resulted reverse-transcription product (2 μL) was used for PCR amplification of specific genes. For primary human keratinocytes, total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). Extracted RNA was used as templates for RT-PCR and qPCR. For RT-PCR, the reverse transcription, initial activating, denaturing, annealing and extension condition of each cycle were 50°C for 30 min, 95°C for 15 min, 94°C for 15 sec, 60°C for 30 sec, 72°C for 30 sec, respectively. The relative expression levels for specific genes was analyzed usng qPCR and the results were calculated using the ΔΔCt method. The mRNA level of each target gene was normalized to the level of GAPDH. The primer sequences for the specific genes are listed in the Supplemental Table 1.