Nasal Respiratory Epithelial Cell Collection and RNA Isolation
Nasal brushings were obtained from the mucosal surface using a 0.8 mm interdental brush (TePe, Ireland). The nasal brush was then transferred immediately to 600 µl lysis buffer (Exiqon, Germany) containing 6 µl β-mercaptoethanol (Sigma-Aldrich, USA) and gently pushed through a sterile pipette tip to release the lysate into the lysis buffer. Brushings from two nostrils were pooled. Total RNA was isolated usingmiRCURYTM RNA Isolation Kit (Exiqon, Germany). Lysate transferred to the lysis buffer was homogenized and after Proteinase K (Thermo Fisher Scientific, USA) treatment, it was transferred to mini spin column (Exiqon, Germany). After DNase I treatment (Bio-Rad), total RNA was eluted in RNase free H2O. Total RNA was stored at -80 0C. RNA quality and quantity was determined using NanoDrop ND-1000 (Thermo Fisher Scientific, USA).