BioProject

Comparison of global gene expression in the proximal and distal colonic epithelium in azoxymethane treated rats. More...

Comparison of global gene expression in the proximal and distal colonic epithelium in azoxymethane treated rats. Keywords: disease state analysis Overall design: Ten male Sprague Dawley rats weighing approximately 245 ± 14.5 g were housed in wire-bottomed caging in a temperature controlled room (22-24°C) with a 12 h light/dark cycle. They were randomly allocated into two groups (n=5) with approximately equal body weights. They were given free access to water and modified AIN-93G diet for 10 days when they were injected with 15 mg of AOM/kg subcutaneously (Sigma Chemical Co., St. Louis, MO, USA). They were anaesthetised with isoflurane and killed by exsanguination six hours after injection. The large bowel (excluding the rectum) was removed, opened longitudinally along the mesenteric border and digesta removed. The colon was rinsed clean with PBS and transferred to a chilled ceramic plate. The most distal and the most proximal 0.5 cm were discarded. Mucosal samples for gene expression and protein analyses were collected by scraping the next 4 cm of proximal and distal colon with new microscope slides. The mucosal scraping was transferred into RNAlater (Sigma Chemical Co., St. Louis, MO, USA) and stored at -80oC for later processing. All instruments were replaced or cleaned thoroughly between animals. All procedures involving animals were approved by the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Human Nutrition Animal Ethics Committee and complied with the Australian code of practice (2004). The distal and proximal colonic mucosal samples were removed from the RNAlater stabilisation reagent (Sigma, Australia) and placed in 1ml of TRIzol® Reagent (Invitrogen, Sydney, N.S.W., Australia). Samples were then homogenised using beads (mix of 2.5mm glass and 0.1 - 1.0mm diameter silicon-zirconian beads) in a MiniBeadbeater-8™ (BioSpec Products Inc. Oklahoma, US). Total RNA was then extracted according to the TRIzol® Reagent manufacturer’s instruction after which samples were further purified using RNAeasy mini spin columns (QIAGEN, Doncaster, Victoria, Australia) with a DNase on-column digestion as per the manufacture’s instructions. The integrity of the RNA was checked using a Bioanalyzer 2100 (Agilent Technologies) and quantified using a NanoDrop® ND-1000 Spectrophotometer. RNA (4.5ug) samples were processed for Microarray expression analysis using high-density oligonucleotide arrays (Affymetrix® GeneChip array, Affymetrix®, Santa Clara, CA, USA) commensurate with the manufacturer’s instructions. Less...