RT-PCR and real-time PCR analysis for MUC5AC mRNA expression

Total RNA was isolated from human NCI-H292 airway epithelial cells and human nasal epithelial cells according to the manufacturer’s instructions (Applied Biosystems). Each sample was reverse transcribed into cDNA using the GeneAmp RNA PCR core kit (Thermo Fisher Scientific, Waltham, MA, USA). PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed on each individual sample as a positive control. Realtime PCR was performed using the LightCycler Fast Start DNA Master SYBR Green kit (Roche, Basel, Switzerland). Data were normalized to GAPDH. The primer sequences, conditions, and expression of the results followed published protocols for MUC 5AC [15,16].