strain: C57Bl/6J gender: male age: 13 weeks tissue: colonic scrapings
Treatment protocol
After 2 weeks mice were switched to semi-synthetic diets, either containing 10 en% of fat (low fat diet; LFD) or 45 en% of fat (high fat diet; HFD). Diets were based on Research Diets formulas D12450B/D12451, with adaptations regarding type of fat (palm oil instead of lard) and carbohydrates to mimic the fatty acid and carbohydrate composition of the average human diet in Western societies. Diets were prepared by Research Diet Services (Wijk bij Duurstede, The Netherlands). After 2 weeks on either the LFD or HFD, the infusion period started. Mice were assigned to one of 4 treatment groups: Control, Acetate, Propionate or Butyrate. On 6 consecutive days, mice were mildly sedated with a mixture of isofluorane (1.5%), nitrous oxide (70%) and oxygen (30%) 2h before the start of the dark phase, where after they received a rectal infusion of the test solutions. At time of infusion, mice were kept under sedation. Mice received an 80 µL saline solution (Control; n = 6 per diet group), or an 80 µL saline solution containing 100 mM sodium acetate (Acetate; n = 6), 100 mM sodium propionate (Propionate; n = 6) or 100 mM sodium butyrate (Butyrate; n = 6). All solutions had a pH of 6.5 and were isotonic. The solutions were administered by inserting a gel loading tip 3 cm into the rectum and slowly pipetting the solution out of the tip. 4 h after the last (6th) infusion, mice were anaesthetized with isoflurane where after the colon was excised. The adhering fat around the colon was carefully removed, and the colon was cut open longitudinally. The intestinal content was removed and the tissue was rinsed with phosphate buffered saline. Subsequently, the epithelial lining of the colon was scraped, snap frozen, and stored at -80 °C for RNA isolation.
Growth protocol
Male C57Bl/6J mice (Charles River, Maastricht, the Netherlands), 8 weeks of age, were individually housed in a light- and temperature-controlled facility (lights on 23:00 to 11:00, 21 °C). Mice were fed a standard laboratory chow (RMH-B, Arie Blok, Woerden, the Netherlands), and had free access to drinking water.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from scrapings using TRIzol reagent, whereafter purified total RNA was isolated using Qiagen RNEasy columns. RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
Label
biotin
Label protocol
One hundred nanogram of RNA was used for whole transcript cDNA synthesis with the Ambion WT expression kit [catalog number 4411974] (Applied Biosystems/Life Technologies, Nieuwekerk a/d IJssel, The Netherlands).
Hybridization protocol
Hybridization and washing of the Affymetrix GeneChip Mouse Gene 1.1 ST peg arrays were performed on a GeneTitan Instrument (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations.
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan instrument (Affymetrix, Santa Clara, CA).
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.24.0).
