strain: C57BL/6 genotype/variation: Caspase-1 null gender: male tissue: jejunum age: 14-16 wks sample type: scraped off epithelial cells, 2hrs after oral lipid load
Treatment protocol
Animals were maintained on regular lab chow. Mice were used at age between 14-16 weeks. For sampling of liver tissue, mice were fasted over night (16hrs). For sampling of small intestine, overnight fasted mice first received an intragastric load of 200 µL olive oil (Carbonell, Cordoba, Spain), and 2 hrs later small intestines were removed. The small intestine was subsequently divided into duodenum, jejunum, and ileum, and the epithelial cell layer was scraped off. All tissue samples were immediately snap-frozen in liquid nitrogen and stored at -80°C for RNA isolation.
Growth protocol
Caspase-1 null mice were backcrossed ten generations to C57Bl/6 mice and age-matched wild-type C57Bl/6J mice were used as control mice. Mice were housed under standard conditions with a 12-hour light-dark cycle and were fed a standard mouse chow diet with free access to water. Experiments were performed in 14 to 16-week old animals.
Extracted molecule
total RNA
Extraction protocol
Total RNA was obtained by extraction with TRIzol reagent (Invitrogen, Carlsbad, CA), followed by DNAse treatment and column purification using the RNeasy mini kit (Qiagen, Hilden, Germany). RNA integrity was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands) with 6000 Nano Chips. RNA was judged as suitable only if samples showed intact bands of 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had a RNA integrity number (RIN) above 8.0.
Label
biotin
Label protocol
The Ambion WT Expression kit (Life Technologies, Carlsbad, CA; P/N 4411974) in conjunction with the Affymetrix GeneChip WT Terminal Labeling kit (Affymetrix, Santa Clara, CA; P/N 900671) was used for the preparation of labeled cDNA from 100ng of total RNA without rRNA reduction.
Hybridization protocol
The Affymetrix GeneChip Mouse Gene 1.1 ST arrays are peg arrays arranged into the standard 96 well plate format. The 24 arrays that were used in this experiment were provided as 1x24 array plate. Array hybridization, washing and scanning were performed on a GeneTitan Instrument according to the manufacturer’s recommendations.
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan instrument.
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.16.0). The SI samples were normalzed together, separately from the liver samples.
