Il10-/- and C57BL/6J control mice were received from The Jackson Laboratories (Bar Harbor, Maine, USA) at four or six weeks of age. All mice were housed singly in shoebox-style cages containing Alpha-Dri bedding material (Shepherd Specialty Papers), a clean sheet of tissue paper, and a plastic tube for environmental enrichment. The animal room was maintained at a temperature of ~22°C and humidity of ~50% with a 12 hour light/dark cycle. All mice had ad libitum access to water, which was refreshed twice a week. Mice were offered fresh food 6 days per week and food intake was estimated at each feeding by weighing food remaining from the previous feeding. The food offered to all groups was adjusted weekly to match the intake of C57BL/6J mice in the control diet group. Mice were weighed three times per week and carefully monitored for disease symptoms (weight loss, soft faeces, inactivity).
Growth protocol
Mice were weighed six times a week throughout the experiment to determine body weight changes. To minimize variation in the time interval between the last food intake and tissue sampling, mice were fasted overnight on the night before sampling. On the morning of sampling, food was returned for two hours, followed by a further two hour fast immediately prior to tissue sampling. Mice were euthanased by CO2 asphyxiation/cervical dislocation. The intestine was removed, cut open lengthwise and flushed with 0.9 % NaCl to remove digesta residues. The proximal half of the colon was cut in three pieces, one for histological evaluation, one for gene expression studies and one backup sample. Samples of the colon tissue for gene expression analysis were frozen in liquid nitrogen and stored at - 80 ºC. Samples for histological analysis were stored in formalin solution (10 % neutral buffered) at room temperature.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the colon tissues using Trizol (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer, with a subsequent purification step using RNeasy columns (Qiagen, San Diego, CA, USA). RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and RNA quality was examined using a RNA 6000 LabChip Kit and a 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA).
Label
Cy3
Label protocol
The Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies Inc., Palo Alto, California, USA) was used to synthesize cDNA and fluorescent cRNA. Labelled cRNA was made on the same day for all sample pools, including the reference sample. cDNA was synthesized from 500 ng of purified total RNA from each pool using T7 promotor primer and Moloney Murine Leukemia Virus Reverse Transcriptase according to the manufacturer’s protocol.
sample type: Pooled RNA from small intestine, colon, kidney, liver and fetuses
Treatment protocol
No treatment prior to tissue collection.
Growth protocol
Healthy adult male Swiss mice were maintained on a 12 h:12 h light:dark cycle and were given food and water ad libitum.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the colon tissues using Trizol (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer, with a subsequent purification step using RNeasy columns (Qiagen, San Diego, CA, USA). RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and RNA quality was examined using a RNA 6000 LabChip Kit and a 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA).
Label
Cy5
Label protocol
The Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies Inc., Palo Alto, California, USA) was used to synthesize cDNA and fluorescent cRNA. Labelled cRNA was made on the same day for all sample pools, including the reference sample. cDNA was synthesized from 500 ng of purified total RNA from each pool using T7 promotor primer and Moloney Murine Leukemia Virus Reverse Transcriptase according to the manufacturer’s protocol.
Hybridization protocol
Microarray hybridization was performed according to a reference design without dye swap. The in situ hybridization kit-plus (Agilent Technologies Inc., Palo Alto, California, USA) was used to hybridize cRNA samples to Agilent Technologies Mouse G4121A - 44k 60mer oligonucleotide arrays. Cy3-labelled cRNA (sample, 0.75 µg) and Cy5-labelled cRNA (reference, 0.75 µg) were hybridised onto the microarray according to the manufacturer’s protocol. After hybridisation the slides were washed following the supplemental procedures for preventing ozone related problems using (in this order) Gene Expression Wash Buffers 1 and 2, Acetonitrile, and Stabilisation and Drying Solution (Agilent Technologies Inc.).
Scan protocol
Immediately after washing, slides were scanned using an Agilent Microarray Scanner (Agilent Technologies Inc.) with the following settings: 5 µm scan resolution, eXtended Dynamic range selected, XDR Hi 100% and XDR Lo 10% selected for both Red and Green channels. Spot alignment and feature extraction was performed using Feature Extract 9.5.1 software (Agilent) using the GE2-v5_95_Feb07 protocol. After scanning, slides were stored at room temperature in airtight containers away from light.
Data processing
Statistical analysis was performed using Linear models for microarray analysis (Limma) from the Bioconductor project. Blank rows, dark corners, negative controls, landing lights, and positive controls were filtered out before normalization. Data were log transformed before analysis and the mean difference between treatments calculated on this scale, resulting in a log ratio for each probe. The normalized values in the database consist of these log ratios. Quality of the microarray data was assessed on diagnostic plots (boxplots and density plots) and spatial images generated from the raw (non-processed) data. MA plots of the microarray data were drawn in order to check that there was no dependence of the log ratio on the intensity for any slide. Intensity ratio values for all microarray spots were normalized using a global loess smoothing procedure to remove the effect of systematic variation in the microarrays and no background correction was necessary due to homogeneous hybridisation. For each experimental comparison, a candidate list of differentially expressed probe sets was generated by calculating a moderated t-statistic for each probe set using the Limma package. The Limma library implements an empirical Bayes approach to assign differential gene expression. The significance of the log ratio for each probe was determined by calculating one modified t-statistic per probe. Probe sets that satisfied the criterion of ≥ 1.5 fold change (FC) with a moderated p < 0.01 were considered to be significantly different. The normalization used for both experiments was a single print tip global loess with slide normalization with no background correction. There was one slide removed from each experiment (C57BL6J_Gold EthylAcetate_Rep4,T117 Slide6_4 and C57BL6J_Green Control_Rep6,T126 Slide9_4);
