*2.2. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)*

In order to investigate the mRNA expression of the different components of the NF-κB activation pathway and fibrotic markers in bronchial fibroblasts, qRT-PCR was performed. Total RNA was extracted from cell pellets using Trizol Reagent (Invitrogen, Waltham, MA, USA, Cat. No. 15596018), according to manufacturer instructions. RNA quality and yield were determined by Nanodrop (Thermo Scientific, Waltham, MA, USA) spectrophotometric measurements. cDNA synthesis was performed from 200 ng of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA, Cat. No. 4368814) in the Veriti Thermal Cycler (Applied Biosystems, Waltham, MA, USA). qRT-PCR reactions were set up using the 5× Hot FirePol EvaGreen qRT-PCR SuperMix (Solis Biodyne, Tartu, Estonia, Cat. No. 08-36-00001) in QuantStudio 3 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The primers used are listed in Table 1. Gene expression was analyzed using the Comparative CT (ΔΔCT) method after normalization to the housekeeping gene 18s RNA. All results are presented as fold expression change compared to non-asthmatic healthy controls in baseline experiments or untreated controls in LPS stimulation experiments.


**Table 1.** List of primer sequences.


**Table 1.** *Cont.*
