@article {van Schadewijkp768, author = {Annemarie van Schadewijk and Emily F.A. van {\textquoteright}t Wout and Jan Stolk and Pieter S. Hiemstra}, title = {A quantitative method for the detection of spliced X-box binding protein-1 (XBP1) mRNA in primary bronchial epithelial cells (PBEC) as a measure of endoplasmatic reticulum (ER) stress}, volume = {38}, number = {Suppl 55}, elocation-id = {p768}, year = {2011}, publisher = {European Respiratory Society}, abstract = {Accumulation of unfolded or misfolded proteins in the ER can cause ER stress, which is increasingly seen in diseases such as cystic fibrosis, alpha-1 antitrypsin deficiency and Alzheimer disease. ER stress leads to the activation of the unfolded protein response (UPR). UPR signaling involves splicing of XBP1 mRNA and leads to the induction of the chaperone protein BiP and the transcription factor CHOP, which is a key-signaling component of ER-stress induced apoptosis. In present studies, XBP1 splicing is frequently used as an important marker for ER-stress and is visualized by gel electrophoresis which is laborious and difficult to quantify. The aim of this study was to develop and validate a quantitative RT-PCR (qPCR) which detects only the spliced form of XBP1 mRNA. We stimulated PBEC with thapsigargin and tunicamycin, both known ER-stress inducers, and performed a qPCR with primers that were designed to recognize only the spliced form of XBP1. We also performed qPCR for CHOP and BiP and correlated this with the spliced XBP1 mRNA to validate the results. The spliced XBP1 PCR product was also confirmed by DNA sequencing. The correlation of XBP1 splicing with the induction of CHOP and BiP was r=0.962 (p\<0.000) and r=0.884 (p\<0.000), respectively. We compared the new method with the visualization of the spliced XBP1 mRNA by gel electrophoresis and we obtained similar results. In conclusion, we have developed a simple and quantitative method for the detection of spliced XBP1 mRNA.This study was supported in part by a grant from the Netherlands Asthma Foundation.}, issn = {0903-1936}, URL = {https://erj.ersjournals.com/content/38/Suppl_55/p768}, eprint = {https://erj.ersjournals.com/content}, journal = {European Respiratory Journal} }