Quantitative Analysis of Transcriptional Profiles

Real-time polymerase chain reaction (qPCR) quantitative analysis was used to evaluate the transcriptional levels of EGFR, IL1β, TNF-a, IL6, STAT3, mTOR, RELA(p65), BCL2, PKI3CA, AKT1, WNT5A, and PTGS2. Total RNA was isolated from HCs exposed to pepsin at variable pH and controls (RNeasy mini kit; Qiagen Inc., Valencia, CA, USA). RNA quality and concentration were assessed by absorption ratios 260/280 nm (>2.0) and 260 nm, respectively (NanoDrop^TM 1000 spectrophotometer; Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, reverse transcription from total RNA (iScript cDNA synthesis kit; Bio-Rad, Hercules, CA, USA) and qPCR analysis (Bio-Rad real-time thermal cycler CFX96^TM; Bio-Rad, Hercules, CA, USA) were performed using specific primers for target genes and reference housekeeping gene, human glyceraldehyde 3-phosphate dehydrogenase (hGAPDH) (QuantiTect Primers Assays; Qiagen Inc., Valencia, CA, USA) (Supplementary Material; Table S1) and iQ^TM SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Our assay was performed on 96-well plates, in triplicates, and data were analyzed by CFX96^TM software [56 (link),58 (link)]. Relative mRNA expression levels were estimated for each target gene compared to the reference control gene (ΔΔCt).