RNA Extraction and qPCR Analysis Protocol

RNA preparation, qPCR, and primer sequencing were performed as previously described [41 (link)]. Total RNA was isolated from tissues extracted from the dorsal lesions of mice using TRI-reagent. The total RNA concentration was determined using a Colibri Microvolume spectrophotometer (Titertek Berthold, Pforzheim, Germany). Further, total RNA was used as a template for cDNA synthesis using a cDNA synthesis kit (Takara Bio, Shiga, Japan). qPCR was performed using SYBR Green I and a LightCycler 96 instrument (Roche, Basel, Switzerland). The cycling conditions were as follows: one cycle of denaturation at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 10  s, annealing at 55 °C for 10  s, and extension at 72 °C for 10  s. The Cq value for each reaction was determined using LightCycler 96 SW1.1 software. The expression levels of the analyzed genes were normalized to that of GAPDH. The primers used are presented in Table 1.