Strain: IL10-/- Gender: male Age: 5 weeks of age at the start of the experiment, 12 weeks of age when tissue samples taken Tissue: colon tissue
Biomaterial provider
AgResearch (Nutrigenomics New Zealand)
Treatment protocol
Animals. Twenty five male IL-10-/- (C57BL/6J background, formal designation B6.129P2-IL-10<tm1Cgn>/J) mice and twenty five male C57BL/6J (C57) control mice were received from The Jackson Laboratory (Bar Harbor, Maine, USA) at approximately five weeks of age. Mice were housed either in pairs or groups of three (5 mice per treatment) in shoebox-style cages containing untreated wood shavings 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. An AIN-76A powdered diet was supplied twice a week, with sufficient provided to meet the daily intake of IL-10-/- mice, as determined in a previous feeding trial (data not shown). The diet for all groups was sterilized by gamma irradiation (Schering-Plough, Wellington, New Zealand) to a level required for SPF conditions, to minimize the possibility of bacterial introduction to the SPF group of animals. All mice were weighed twice weekly and carefully monitored for disease symptoms (weight loss, soft faeces and inactivity). Experimental design. Both IL-10-/- and C57 mice were randomly divided into five treatment groups with five animals per group. 1. SPF. Housed in Specific Pathogen Free (SPF) conditions (isolator cages supplied with HEPA filtered air (Tecniplast SpA, Buguggiate, Italy)). 2. C. Maintained under conventional conditions. 3. EF. Maintained under conventional conditions and orally inoculated (200 microlitres) with a solutions containing 12 pure strains of Enterococcus (1.2 x 108 colony forming units (CFU)). 4. CIF. Maintained under conventional conditions and orally inoculated with conventional intestinal flora (CIF) derived from healthy age-matched C57BL/6 mice which had been raised under conventional conditions. The CIF inoculation protocol was included to better mimic the complete microbiota associated with the mouse gastrointestinal tract. 5. EF.CIF. Maintained under conventional conditions and orally inoculated with a combination of the EF and CIF inoculation solutions (6.0 x 107 CFU from the EF inoculum). Preparation of EF bacterial solutions for oral inoculation. Seventy two hours prior to inoculation, colonies of the E. faecalis and E. faecium strains were sub-cultured from slope tubes onto fresh Slanetz & Bartley medium (Oxoid, Hampshire, UK), and incubated at 42°C for 48 h. A single colony from each culture was subsequently transferred to 5 mL of Todd Hewitt Broth (Oxoid, Hampshire, UK), and incubated at 37°C for 24 h. Each Enterococcus culture was centrifuged to pellet the bacterial cells (3,000 g, 10 min, 4°C), which were then re-suspended in 5 mL sterile PBS (pH 7.4) and pooled (EF inoculum). Preparation of CIF bacterial solutions for oral inoculation. The healthy, age-matched C57BL/6 mice from which flora were being collected were humanely euthanized by CO2 asphyxiation and cervical dislocation and the gastrointestinal tract (from stomach to just below the caecum) removed. Digesta were collected from the intestine and caecum by gently washing with sterile PBS, pH 7.4, then suspended in a total of 30 mL PBS. After mixing by gentle inversion and a settling period of approximately 5 min, the suspension was collected (CIF inoculum). Inoculation solutions EF and CIF were mixed in a 1:1 (v/v) solution to obtain the EF.CIF inoculum.
Growth protocol
Mice were weighed three 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. Blood was collected from euthanised mice by cardiac puncture. Plasma was separated from red blood cells by centrifugation (2000 g, 10 min, 4 ºC), frozen in liquid nitrogen and stored at - 80 ºC. 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 two pieces, one for histological evaluation and other for gene expression studies. 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. Cy3 (PerkinElmer, Waltham, Massachusetts 02451, USA) was used to label sample groups, while the reference RNA was labeled with Cy5 (PerkinElmer, Waltham, Massachusetts 02451, USA).
Strain: IL10-/- and C57BL/6J Gender: male Age: 12 weeks Tissue: colon tissue Reference sample consisted of RNA extracted from colon tissue of all mice in this experiment (both IL10-/- and C57BL/6J) pooled in equimolar amounts.
Biomaterial provider
AgResearch (Nutrigenomics New Zealand)
Treatment protocol
As per Channel 1
Growth protocol
As per Channel 1
Extracted molecule
total RNA
Extraction protocol
As per Channel 1
Label
Cy5
Label protocol
As per Channel 1
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. Following hybridisation, slides were washed in solutions I, II and III (Agilent Technologies, Santa Clara, CA, USA) and air-dried.
Scan protocol
Slides were scanned using a GenePix 4200A scanner (Molecular Devices Corporation, Sunnyvale, CA, USA) at a photomultiplier tube (PMT) setting of 450 V. Spot identification and quantification were performed using GenePix 6.0 software (Molecular Devices Corporation, 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.
Description
IL10_Colon_Conventional_Rep1_(Pool7)
Data processing
Statistical analysis was performed using linear models for microarray analysis (limma) within the Bioconductor framework. Before analysis, poor quality spots were manually flagged and filtered out. Quality of the microarray data was assessed on diagnostic plots (boxplots and density plots) and spatial images generated from the raw (non-processed) data. All twenty arrays passed these strict criteria and were included in the analyses. Intensity ratio values for all microarray spots were normalized using a within-slide global Locally Weighted Scatterplot Smoothing (Loess) procedure to remove the effect of systematic variation in the microarrays; no background correction was necessary due to homogeneous hybridization. The normalized data from the arrays of each treatment group were averaged. For each comparison, differentially expressed genes were identified using false discovery rate (FDR) control with a threshold of q < 0.05. 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. 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. The significance of the log ratio for each probe was determined by calculating one modified t-statistic per probe using an empirical Bayes approach.
The probability values were then corrected for multiple testing using the Benjamini and Hochberg correction, and a false discovery rate (FDR) calculated. Probes that had an FDR of less than 5 % (q<0.05) were considered to be differentially expressed between treatment.
