Unknown,Transcriptomics,Genomics,Proteomics

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Gene Expression in Rats Fed a Western Diet

ABSTRACT: Resistant starches (RS), fed as high amylose maize starch (HAMS) or butyrylated HAMS (HAMSB), oppose dietary protein-induced colonocyte DNA damage in rats. In this study, rats were fed diets high in fat (19%) and protein (20%) with different forms of digestible starch (low amylose maize starch (LAMS) or low amylose whole wheat (LAW)) or RS (HAMS, HAMSB, or a whole high amylose wheat (HAW) generated by RNA interference (RNAi)) for 11 wk. A control diet contained 7% fat, 13% protein and LAMS. The aim of this study was to detect changes in the expression of DNA damage and repair genes in response to the above dietary treatments. Distal colon tissues from Sprague Dawley rats fed a variety of diets (see summary) were removed from RNAlater stabilisation reagent (Sigma, Australia) they had been stored in,, placed in 1 ml of TRIzol® Reagent (Invitrogen, Australia) and homogenised usingbeads (mix of 2.5 mm glass and 0.1 - 1.0 mm diameter silicon-zirconian beads) in a MiniBeadbeater-8 (BioSpec Products Inc., USA). Total RNA was extracted (using TRIzol® Reagent manufacturer's instructions) and further purified using RNAeasy mini spin columns (QIAGEN, Australia) with a DNase on-column digestion as per manufacturer's instructions.RNA integrity was checked using a Bioanalyzer 2100 (Agilent Technologies, USA) andquantified using a NanoDrop® ND-1000 Spectrophotometer (Thermo Fisher Scientific, USA). RNA samples with insufficinet quality and quantity were not assayed. The numbers of rats arrayed from each diet were as follows: CON, 5;LAMS, 7; HAMS, 8; HAMSB, 9; LAW, 6; HAW , 8. This gives a total of 43 arrays.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Robert Dunne

PROVIDER: E-GEOD-32312 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Comparison of global gene expression in the proximal and distal colonic epithelium in azoxymethane treated rats. Experiment Overall Design: Ten male Sprague Dawley rats weighing approximately 245 Â± 14.5 g were housed in wire-bottomed caging in a temperature controlled room (22-24Â°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. Experiment Overall Design: 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). Experiment Overall Design: The distal and proximal colonic mucosal samples were removed from the RNAlater stabilisation reagent (Sigma, Australia) and placed in 1ml of TRIzolÂ® 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-8â¢ (BioSpec Products Inc. Oklahoma, US). Total RNA was then extracted according to the TRIzolÂ® Reagent manufacturerâs 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 manufactureâs instructions. The integrity of the RNA was checked using a Bioanalyzer 2100 (Agilent Technologies) and quantified using a NanoDropÂ® ND-1000 Spectrophotometer. Experiment Overall Design: RNA (4.5ug) samples were processed for Microarray expression analysis using high-density oligonucleotide arrays (AffymetrixÂ® GeneChip array, AffymetrixÂ®, Santa Clara, CA, USA) commensurate with the manufacturerâs instructions.

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Gene Expression in Rats Fed a Western Diet

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