Quantitative PCR Analysis of Gene Expression

PCR primers obtained from the qPrimerDepot database^{55 (link)} or designed with Primer Designing Tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) were chosen overlapping an intron or hybridizing an exon junction when possible and checked for in silico specificity before synthesis by Eurofins Operon (Supplementary Table S1). qPCR analysis was done in triplicate 15-µL reactions with SYBR Premix Ex Taq Tli RNase H Plus mix (Takara) including 5 µL of 30-fold diluted cDNA and 0.5 µM of each primer. A 2-steps PCR program with hybridization at 61 °C followed by specificity check on fusion curves, was performed on CFX384 thermal cycler (Bio-Rad Laboratories). Quantitative values were normalized using the NORMA-Gene method^{56 (link)} for whole-skin data and with GAPDH and ACTB housekeeping genes for microdissection data^{57 (link)}. Relative expression ratios were calculated by the delta-delta Cq method^{58 (link)}, based on a theoretical efficiency considered at 100%, using as calibrator the untreated condition of similar duration as treated samples (day 10).