Validation of Fibrogenesis and Graft Tolerance Targets

In order to validate some of the specific targets identified by prediction analysis, we decided to concentrate further on processes of lung fibrogenesis and graft tolerance, such as STAT3, SMAD3 and SMAD4 [24 (link),25 (link),26 (link),27 (link)]. RNA was isolated from PBMCs derived from 4 BOS patients, prior to the start and after 6 months of ECP treatment, by miRNeasy Mini Kit (Qiagen). MiR-155-5p and miR-23b-3p expression levels were evaluated as described above. To evaluate STAT3, SMAD3 and SMAD4 gene expression, cDNA was retro-transcribed from 1 µg of total RNA using LunaScript RT SuperMix Kit (NEB). Relative levels of STAT3, SMAD3 and SMAD4 mRNA were assessed using SYBR^® Green Luna^® Universal qPCR Master Mix (NEB) and normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Each experimental condition was performed in triplicate. Relative gene expression level quantification was compared with internal standards and analyzed using the 2^−ΔΔCt method.