Gene Expression Analysis in Tissue Samples

Upon harvest, tissues (skin and lung) were immersed in RNAlater (QIAGEN) and stored at −80°C. The samples were homogenized with the Bullet Blender 24 Gold (Next Advance). RNA was isolated using the RNeasy Micro Kit or RNeasy Fibrous Mini Kit (QIAGEN), and cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific). qPCR was performed using SYBR GreenER qPCR SuperMix (Invitrogen, Thermo Fisher Scientific), and quantification of gene expression was performed as previously described (47 (link)). All samples were normalized to YWHAZ gene expression, and results are expressed as the fold change of C_t values (mean of 3 replicates) compared with controls, using the 2^-ΔΔCt formula.