strain: IL10-/- mice (B6.129P2-IL10<tm1Cgn>/J) background strain: C57 mice (C57BL/6J), control gender: male age: 5 weeks tissue: colon tissue
Biomaterial provider
The Jackson Laboratory, Bar Harbor, Maine, USA
Treatment protocol
A total of 30 male IL10-/- (B6.129P2-Il10<tm1Cgn>/J) and 30 male C57 control (C57BL/6J) mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). All mice were between 28 and 35 days of age at the start of the study. For convenience and consistency in reporting, their age was defined as 35 days or 5 weeks of age. The mice were housed individually in standard shoebox size cages containing untreated wood shavings and maintained in an air-conditioned animal room with a 12-h light-dark cycle. Animals had free access to water and were fed an AIN-76A standard powder diet prepared in-house. This study was approved by the AgResearch Ruakura Animal Ethics Committee in Hamilton, New Zealand according to the Animal Protection Act (1960) and Animal Protection Regulations (1987) and amendments. Four days after arrival, all mice were inoculated orally with a mixture of Enterococcus faecalis strains and bacteria commonly found in the intestinal lumen to obtain a more consistent and reproducible intestinal inflammation. Mice were randomly assigned to five sampling groups (7, 8.5, 10, 12 and 14 weeks of age).
Growth protocol
Bodyweight was determined three times a week and the mice carefully monitored for disease symptoms (weight loss, soft faeces, inactivity, etc.). Throughout the experimental period, dietary intake was estimated and adjusted daily to equal the mean amount of food consumed by IL10-/- mice on the previous day to ensure similar intakes between control (C57) and IL10-/- mice. On the day prior to sampling, mice were subjected to a fast-feed period prior to sampling. The mice were euthanased by CO2 asphyxiation and cervical dislocation. The intestine was removed, cut open lengthwise and rinsed with 0.9% NaCl to remove traces of digesta. The intestine was divided into duodenum, jejunum, ileum and colon. One piece of each intact tissue was stored at room temperature in 10% phosphate buffered formaldehyde for histological analysis, another immediately frozen in liquid nitrogen and kept at −85°C for genomic analysis. Plasma was separated from the blood, frozen in liquid nitrogen and stored at −85°C for biochemical analysis.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from homogenized full-thickness colonic tissues in TRIzol reagent (Invitrogen, Auckland, NZ) and purified with RNeasy columns (QIAGEN, San Diego, CA, USA). Quantity and integrity of RNA were assessed by a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and an Agilent 2100 bioanalyser (Agilent Technologies, Santa Clara, CA, USA), respectively. Samples with a 28s/18s peak ratio ~2.0 and a RNA integrity number >8 were considered for microarray hybridization and quantitative real-time PCR. For the comparison of gene expression levels, colonic RNA of IL10-/- and C57 at 7 and 12 weeks of age were compared because of a low HIS variability in the colon within those time points. A reference design with 15 arrays was used, where each individual RNA sample was hybridized with a reference sample onto the array, in a total of 3 or 4 biological replicates per treatment. Reference RNA was prepared by pooling equal amounts of purified total RNA extracted from small intestine, colon, kidney, liver and fetuses from normal, healthy Swiss mice.
Label
Cy3
Label protocol
Amplification and labeling was processed according to Agilent Technologies protocols. Briefly, cDNA and cRNA were synthesised using the Low RNA Input Linear Amplification kit (Agilent Technologies). cRNA was labelled with cyanine 3-CTP (samples) and cyanine 5-CTP (reference) dyes (10mM; Perkin-Elmer/NEN Life Science (NEL 580, 581), Boston, MA, USA) and the dye incorporation monitored spectrophotometrically using a Nanodrop ND-1000.
sample type: Pooled RNA from small intestine, colon, kidney, liver and fetuses from normal, healthy Swiss mice
Biomaterial provider
Crop & Food Research (NZ) Small Animal Unit
Treatment protocol
Reference
Growth protocol
Reference
Extracted molecule
total RNA
Extraction protocol
as for channel 1
Label
Cy5
Label protocol
As for channel 1 (but Cy5)
Hybridization protocol
Labelled cRNA was hybridized on an Agilent Technologies 44k (G4122-60510) mouse 60mer oligonucleotide array (containing 44,290 probe sets which represent 33,994 unique genes) using the in situ hybridization plus kit (Agilent Technologies) and following the manufacturer’s protocols.
Scan protocol
Arrays were scanned using a GenePix Professional 4200A scanner (Molecular Devices, Sunnyvale, CA, USA). Spot identification and quantification was performed using GenePix 6.0 software (Molecular Devices). All slides were individually checked and manually flagged for abnormalities. probe sets quantified. There are low, medium and high scan data available.
Description
This data relates to a submitted paper "Transcriptomic and proteomic analyses to characterize onset and progression of colon inflammation in interleukin-10 gene-deficient mice" Further details :
The formaldehyde-fixed intestinal tissues were embedded in a paraffin block, cut into 5 µm thick sections and stained with haematoxylin and eosin. The stained sections were scored by a pathologist (who had no knowledge of the experiment protocol) for the following aspects of inflammation: inflammatory cell infiltration (monocytes, neutrophils, fibrin exudation and lymphangiectasis), tissue destruction (enterocyte loss, ballooning degeneration, oedema and mucosal atrophy) and tissue repair (hyperplasia, angiogenesis, granulomas and fibrosis). A rating score between 0 (no change from normal tissue section) and 3 (lesions involving most areas and all layers of the tissue section including mucosa, muscle and omental fat) was given for each aspect of inflammatory cell infiltration, tissue destruction, and tissue repair. For each intestinal section, the histological injury score (HIS) was calculated as: HIS = (inflammatory lesions score) × 2 + tissue destruction score + tissue repair score. Inflammatory cell infiltration represented the main characteristic of the observed inflammation, therefore the sum of inflammatory lesions was multiplied by 2 to give more weight to this value. The concentration of serum amyloid A (SAA) was measured in plasma to complement the histology scores using a murine specific SAA kit (Tridelta Development Limited, Maynooth, County Kildare, Ireland), according to the manufacturer’s instructions.
Data processing
Statistical analysis and quality assessment of the microarray data were performed using linear models for microarray analysis (Limma) within the Bioconductor framework [Smyth 2005]. Data quality was assessed on diagnostic plots (boxplots and density plots) and spatial images generated from the raw (non-processed) data. All arrays passed the quality control and were included in the analyses. Medium scan data were used for normalization. Intensity ratios 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 hybridization [Smyth and Speed 2003].
The normalized data from the arrays of each time point group were averaged. For each comparison of interest (IL10-/- 7 vs. C57 7 and IL10-/- 12 vs. C57 12), a list of differentially expressed genes was generated by calculating moderated t-statistics and false discovery rate using the Limma package, which implements an empirical Bayes approach to assign differential gene expression.
Probes from the microarray dataset that satisfied the cut-off criteria of a 1.5-fold change (FC) in expression (up- or down-regulated) and a FDR (q-value) less than 0.05 were considered significantly different and used for pathway and functional analysis.
