Human RNA Isolation from Tempus Tubes and quantitative Real-Time PCR.
At Baseline visit whole blood was collected in Tempus® Blood RNA Tubes (Life Technologies™, GmbH, Darmstadt, Germany) and RNA was extracted with the MagMax for Stabilized Blood Tubes RNA Isolation Kit. Synthesis of cDNA and consequently real time-PCR were performed as described for murine cells below with the following primers andsequences: hHPRT (5´-TGA CAC TGG CAA AAC AAT GCA-3´, 5´-GGT CCT TTT CAC CAG CAA GCT-3´), PD-1 (fw: 5´-CAG TTC CAA ACC CTG GTG GT- 3´; rev: 5´-GGC TCC TAT TGT CCC TCG TG- 3´).
FEV1 .
The Spirometry method for the lung function analysis was performed by using a Vyaire Bodyplethysmograph (Chicago,USA). ERS Criteria were followed during the study. The quality control was performed by an experienced technician and a physician and supported by the software.FEV1 (=Forced expiratory volume in 1 second) and FVC (=Forced vital capacity) were measured at Baseline visit (B0) by using spirometry. After a period of normal breathing the participant should inhale maximal, directly followed by maximal and fast exhalation. The volume exhaled in one second is FEV1. The total exhaled volume is FVC. The ratio FVC/FEV1 is stated as FEV1%.