Peyer’s patches were removed from the intestinal segments which were then dissected longitudinally and digested in a Trypsin (0.25%)/EDTA (0.02%) solution (Gibco BRL) for 30 minutes shaking at 37C. The cell suspension was then centrifuged (200g x 10 min), resuspended in DMEM (10% FCS). The small intestinal digest prepared was filtered and incubated with FITC-CD45 antibody. The sample was incubated with anti-FITC microbeads and MACS column, as described above, with the epithelial population being isolated by negative selection. This epithelial population was denoted as the villus-associated epithelium (VAE).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted and DNAse-I treated using the Absolutely RNA RT-PCR Miniprep kit (Stratagene, LaJolla, CA, USA) following the procedure followed provided by the manufacturer. The total RNA samples were then analyzed by electrophoresis in a 1.5% 1X TBE agarose gel (Sigma) and visualized by ethidium bromide (Sigma) staining under UV light. Total RNA was quantified using a GeneQuant II spectrophotometer (Amersham-Pharmacia, Buckinghamshire, UK). Absorbance readings were taken at 260 nm and 280 nm and the concentration and the ratios determined.
Label
33P
Label protocol
2 g of the total RNA isolated from the M cell, FAE, VAE and PPL samples were reverse transcribed using the R&D Systems cDNA labeling kit (R&D Systems, Oxon, UK) in the presence of [-33P]-dCTP (Amersham Life Science). Briefly, the RNA and mouse cytokine-specific primers (provided with kit) were first denatured at 90C for 2 minutes, incubated at 42C for 20 minutes and then the reagents for reverse transcription were added. Each reverse transcriptase (RT) reaction contained RT buffer, dNTP mix (333 M dATP, dGTP, dTTP, 1.67 M dCTP), 20U/ml of RNasin (Promega, Southampton, UK), 50 U AMV reverse transcriptase and [-33P]-dCTP, 20 Ci, 2000-3,000 Ci/mmol. The RT reaction was allowed to proceed for 2 Hours at 42C. Removal of unincorporated nucleotide was achieved by the use of Sephadex G-25 spin column.
Hybridization protocol
The 33P cDNA probes from the samples of interest were hybridized overnight at 65C to the Mouse Cytokine Expression Array Version 1.0 (Sigma-R&D Systems). The cDNA arrays were then washed three times with 0.5X SSPE/1%SDS (low stringency) and twice at 65C with 0.1 X SSPE/1%SDS (high stringency). The arrays were then exposed to a phosphorimaging screen (Molecular Dynamics, Amersham Biosciences, UK) overnight. The screens were then scanned at 50-m pixel size using a STORM phosphorimager (Molecular Dynamics).
Scan protocol
The screens were then scanned at 50-m pixel size using a STORM phosphorimager (Molecular Dynamics).
Description
Phoretix Array2 Software (Nonlinear Dynamics, Newcastle-upon-Tyne, UK) was used to analyze the array images. The images were background subtracted and the intensity normalized to negative controls and to the selected housekeeping gene, beta-actin. The normalized data was then imported to Microsoft Excel (Microsoft, Ireland), where the ratios of differences between the samples of interest were calculated. The normalized data was also analyzed using Cluster and TreeView (Michael Eisen, Stanford University, USA). Using Cluster, the array data was filtered; log transformed, median centered and hierarchically clustered and visualized using TreeView. The Cluster transformed data was also analyzed using J-Express software (MolMine AS, Bergen, Norway) to evaluate common gene expression trends. Gene lists were complied and the function of the genes of interest were determined using Genecards (http://bioinformatics.weizmann.ac.il/cards-bin/carddisp?[gene) and the PubMed database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed).
Data processing
The normalized data was then imported to Microsoft Excel (Microsoft, Ireland), where the ratios of differences between the samples of interest were calculated. The normalized data was also analyzed using Cluster and TreeView (Michael Eisen, Stanford University, USA). Using Cluster, the array data was filtered; log transformed, median centered and hierarchically clustered and visualized using TreeView. The Cluster transformed data was also analyzed using J-Express software (MolMine AS, Bergen, Norway) to evaluate common gene expression trends. Gene lists were complied and the function of the genes of interest were determined using Genecards (http://bioinformatics.weizmann.ac.il/cards-bin/carddisp?[gene) and the PubMed database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed).
