Influence of isolation methods on gene expression profiles.

<p>A) Differentially regulated genes in IEC isolated with EDTA/DTT compared to enzymatic release. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3^rd and 97^th percentile of the data. Significance testing was performed using paired Students T-test and values for <i>p</i><0.05 were considered to be statistically significant. B) Cluster analysis of all analyzed patients. Individuals are numbered. An L signifies IEC isolated using enzymatic release. An E signifies IEC isolated using the EDTA/DTT-based approach. All six patients were included in this analysis. Tissue samples for EDTA/DTT and enzymatic release were obtained from the same side in each patient and all analyses were performed in triplicate.</p

Cytokeratin 8 and CD3γ copy numbers in intestinal epithelial cells.

<p>A) FACS analysis of T cell contamination. Positive selection of IEC was performed using CD326 Microbeads followed by staining using T-cell receptor antibodies and Pacific Blue Annexin V to detect apoptotic cells. Panel A1 shows an example of IEC isolated using the enzyme based protocol, panel A2 using the EDTA/DTT based protocol. T-cell receptor positive cells are located in quadrant Q1 and Q2. Analysis of absolute mRNA copy numbers of cytokeratin 8 and CD3γ in intestinal epithelial cells separated by positive selection using CD326 Microbeads cells. B) Expression of mRNA copy numbers of Cytokeratin 8 and CD3γ. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3^rd and 97^th percentile of the data. Significance testing was performed using paired Students T-test and values for <i>p</i><0.05 were considered to be statistically significant.</p

Analysis of cytosine methylation of promoter regions of selected genes.

<p>A) CpG methylation of promoter regions of selected genes analysed by MeDIP. B) CpG methylation of promoter regions of selected genes analysed by pyrosequencing. Data is expressed as box and whisker plots. The length of the boxes represents the interquartile range and the whiskers the 3^rd and 97^th percentile of the data. Significance testing was performed using paired Students T-test and values for <i>p</i><0.05 were considered to be statistically significant.</p

DNA methylation of EPCAM in whole biopsy samples compared to separated epithelium and negative cell fraction.

<p>A) DNA methylation of 5 CpGs within the EPCAM gene using a pyrosequencing assay. B) Total CpG methylation comparing EPCAM versus NOD1. Assays included 5 CpG sites for EPCAM and 2 for NOD1. C) EPCAM mRNA expression in whole biopsies compared to epithelial positive and negative separated cell fraction. Data is expressed as mean +/− SEM of 5 tissue samples. Differences were considered as statistically significant if p<0.05 (** p<0.001, ***p<0.0001).</p