RNA-Seq and proteomics of Crohn's disease: Terminal ileum of inflamed and non inflamed paired tissue biopsy

The cause of chronic inflammation manifestations such as inflammatory bowel disease (IBD) is still not yet fully understood. Evidence however points to the convergence of environmental factors, genetic factors, intestinal microbiota and the immune response at the intestinal epithelial surface that consequently leads to a breakdown of barrier function in IBD. An increasing number of theories implicate a dysfunctional intestinal epithelial surface in the pathogenesis of IBD and progression to complicated disease, which contributes significantly to the IBD burden. As yet, the significance of the intestinal epithelial surface in IBD manifestation and progression has not been explored using a systems biology approach, that compares the mucosa of healthy patients to those with IBD. In the context of dyregulation of the epithelial barrier, there is a scarcity of publications that explore the contribution of proteins- the effectors of the cells and RNA message in the pathways affected by CD and..., Collection of samples
Pinch biopsies from Crohns disease patients undergoing colonoscopy at Concord Repatriation General Hospital were collected from inflamed Terminal ileum portion of bowel and the closest macroscopically normal mucosa section. Two pinches were collected from both the inflamed (I) and non-inflamed (NI) portions of bowel. Pinch biopsies were immediately placed in 150μl of RNAlater (ThermoFisher Scientific, Melbourne, Australia) and stored at -80°C until use. Bowel was graded as either I or NI by the gastroenterologist performing the colonoscopy based on the SES-CD. In addition, samples were also collected from healthy control patients undergoing colonoscopy for unrelated conditions.
Extraction of total RNA
7–20mg of frozen bowel tissue was added to a chilled and washed 2ml Eppendorf tube. Lysis buffer from the Qiagen RNAeasy Mini Kit (Melbourne, Victoria, Australia) was added along with 6mg of 1.0mm zirconium beads previously washed with IPGphor detergent and nitric a...,