strain: C57Bl/6J gender: male age: 16-17 weeks tissue: liver treatment: infused with 0.3 M sodium [2-13C]propionate
Treatment protocol
Mice were equipped with a permanent cecum catheter and allowed a recovery period of at least 5 days. Cecal cannulas were flushed daily with phosphate buffered saline. On the day of the experiment, mice were individually housed and fasted from 6:00 to 10:00 a.m. All infusion experiments were performed in conscious, unrestrained mice. Four different groups received either a control phosphate-buffered saline solution, a 0.3 M sodium [1-13C]acetate (99 atom %, Sigma-Aldrich) solution, a 0.3 M sodium [2-13C]propionate (99 atom %, Sigma-Aldrich) solution or a 0.3 M sodium [2,4-13C2]butyrate (99 atom %, Sigma-Aldrich) solution infused via the cecum catheter at an infusion rate of 0.2 ml/h. The resulting rate of infusion of labeled SCFA was 2.4 mmol/h/kg. After 6 h of infusion, animals were killed by cardiac puncture under isoflurane anesthesia. Livers were removed, freeze-clamped, and stored at -80 °C for RNA isolation.
Growth protocol
Male C57Bl/6J mice (Charles River, L’Arbresle Cedex, France), 2 months of age, were housed in a light- and temperature-controlled facility (lights on 6:30 a.m. to 6:30 p.m., 21 °C). Mice were fed a semi-synthetic diet based on Research Diets’ formulations D12450B, with adaptations regarding type of fat (palm oil instead of lard) and carbohydrates, for 6 weeks and had free access to drinking water.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from livers 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). For array analysis, RNA of 4-8 mice per group was subsequently pooled at a concentration of 500 ng/mouse.
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' (v1.22.0).
