Aminoallyl modified nucleotides were coupled with CyDye selleck chemicals llc using the Post-Labeling Reactive Dye kit (Amersham Biosciences, Little Chalfont, UK). The MITChip microarrays were produced by Agilent Technologies (Agilent Technologies, Palo Alto, CA, USA). Each array was hybridized with two samples, labeled

with Cy3 and Cy5, respectively. Combined Cy3- and Cy5-labelled target mixtures were fragmented by adding 1 μL of Ambion 10× fragmentation reagent (Ambion Inc.), and incubation at 70°C for 20 min, according to the manufacturer’s instruction. Fragmentation was stopped by adding 1 μL of Ambion stop solution. Hybridization mix was prepared by adding to the RNA mixture 31.5 μL of 20× SSC, 6.3 μL of 10% SDS, 25 μL of Agilent Control Target mix and RNAse-free water to a total volume of 210 μL. Hybridization was carried out at 62.5°C in a rotation oven (Agilent) for 16 h. Slides were washed at room temperature in 2× SSC, 0.3% SDS for 10 min, and at 38°C in 0.1× SSC, 0.3% SDS for 10 min. SDS was completely removed by washing the slides in 0.06× SSPE for 5 min, followed by a quick dry with compressed nitrogen. Data were extracted from microarray images using the Agilent Feature Extraction software, version 9.1 (http://www.agilent.com). Data normalization and the further microarray analysis were performed using a set of R-based scripts (http://www.r-project.org) in combination

with a custom designed relational database which runs under the MySQL database management system (http://www.mysql.com) [51]. In PLX-4720 order to relate the change of the microbiota to sampling site, environmental variables or genotypes, multivariate analysis was performed by RDA as implemented in the CANOCO 4.5 software package (Biometris, Wageningen, The Netherlands)

on average signal intensities for 99 bacterial groups (level 2). All environmental variables were transformed as log(1 + X). A Monte Carlo permutation test based on 999 random permutations was used to test the significance. p-values <0.05 were considered significant. Diversity of microbial profiles obtained by MITChip analysis was expressed as Simpson's reciprocal index of diversity (1/D). Diversity was calculated with the equation l = 1/ΣPi2, where Pi is the proportion of taxon i, that is, the proportion of each probe signal compared to the total signal for each sample. A higher Simpson's index value indicates a higher Oxaprozin degree of diversity. Male, 9- to 10-week-old mice were stratified from litters but randomly picked and placed in pairs in clean cages. Acute colitis was induced by DSS, MW 36,000–50,000 kD (MP Biomedicals, Illkirch, France) 1.5% w/v in drinking water for 7 days and mice where further observed through a recovery period of 7 days on regular drinking water. Mice were weighed and inspected every 24 h−1 for anal blood and for diarrhea (def.: complete moisture of fur between anus and tail root). In indicated experiments, mice were provided with drinking water containing 2.