Treg_Chip_Expression_Profile_Healthy_vs_Crohns_Disease

Regulatory T cells from patients with Crohn´s disease show an inadequate increase of their frequency in inflamed mucosa. Using a self-developed mRNA-array (Treg Chip), we wanted to identified candidate genes for a disturbed Treg migration Keywords: cell type comparison To obtain individual transcription profiles, we isolated CD4+CD25+ and CD4+CD25- T cells from peripheral blood of 6 patients with Crohn´s disease (3 patients each with active (aCD) and inactive disease (iCD)) and 11 healthy control (HC) using magnetic cell sorting. Micoarray analysis was done using our self-developped human TReg Chip. RNA from CD4+CD25+ T cells was isolated. After ethanol precipitation, quality and integrity of the total RNA was controlled. Samples were prepared by applying a double-linear amplification method. Briefly, the first round of RNA-amplification was performed without biotinylated nucleotides. After clean-up of the precipitated aRNA synthesis of second round, first-strand cDNA. Subsequent second-strand cDNA was prepared like in the first round but integrating an additional RNAse H incubation step to digest the aRNA before annealing of the T7T23V primer. The second round of RNA amplification was performed as an in vitro transcription assay in the presence of biotinylated UTP. The concentration of the obtained biotin-labelled cRNA was determined by UV absorbance and its quality as means of product length distribution was again checked. In all cases, 15 µg of each biotinylated cRNA preparation were fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre). Samples were hybridized to individual Human TReg Chips for 16 hours at 42°C. After hybridisation the microarrays were washed, stained with Cy5-streptavidin and read . Signal intensities were qualified and quantified by means. Spots of poor quality (flag = 3) were excluded from further analysis. To adjust arrays from different experiments, data normalisation based on median signal intensities of the housekeeping genes was carried out. In addition, to adjust the data set from HC (11 donors, 29 chips) to aCD or iCD (3 patients each, 6 chips), median normalised signal intensity for each gene and donor was calculated (MV), standard derivation (SD) was assigned and normalised (CV = SD/MV). Statistical Analysis of Microarrays (SAM) was used to ascertain gene expression changes in CD4+CD25+ regulatory and CD4+CD25- naïve T cells. For this, only genes with a CV < 0.3 in 8/11 HC were evaluated.