Transcriptional Analysis of MHPC

We isolated total RNA (RNeasy mini kit; Qiagen Inc., CA, USA) from MHPC exposed to acidic bile alone, pre-treated and post-treated with BAY 11-7082 groups, and controls, to evaluate the transcriptional levels of RELA (p65), bcl-2, TNF-α, EGFR, STAT3, WNT5A and IL-6, using quantitative real time polymerase chain reaction (qPCR) analysis and specific primers for mouse genome, as previously described [8 (link), 10 (link)]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference housekeeping gene (QuantiTect Primers Assays; Qiagen) [10 (link)]. We performed assays in 96-well plates, in triplicate for each sample, and data were analyzed by CFX96™ software. Relative mRNA expression levels were estimated for each target gene relative to reference gene (ΔΔCt). (Data were obtained from three independent experiments).