The median values of triplets were used to calculate relative expression of each gene according to the ΔΔCt method [50]. Unsedated animals were held in the hands of the researchers and allowed to defecate directly into

a clean tube. Mice were then sacrificed and dissected as described above. The cecum was amputated in the ileocecal junction and at the proximal end of the colon and the distal third Osimertinib cost cut transversally and collected. The distal two-thirds of the cecum were then cut open longitudinally and luminal contents were gently collected with a disposable spatula. The rest of the luminal contents was washed off the cecum biopsy in three consecutive baths of cold PBS before the biopsy was laid flat with its mucosal side up and the mucosa was scraped off with a disposable rubber cell scrape and collected. All

collected samples were put straight in clean 1.5 mL Eppendorf tubes (Sarstedt, Nümbrect, Germany), snap frozen in liquid nitrogen and kept at −70°C until use. Design of the MITChip, sample processing, and data analysis was performed as described for the HITChip [51]. Briefly, HTS assay 90,000 sequences derived from the mouse intestinal tract were obtained from ARB-Silva database. Design of OTUs was performed with a cutoff of 98% and a total of 1885 OTUs were used for designing probes. Both V1 and V6 regions were exported and divided into three overlapping 24 nucleotide parts. Calculation of the predicted melting temperature (Tm) of the probes was achieved using the SantaLucia algorithm. The sequences of the probes were optimized so that the Tm fitted into a selected 5°C range (60–65°C). Before optimization of the probes, 30% of the probes fitted in the selected region of Tm, while after optimization, this percentage increased to 98%. Redundant probes were screened, and unique probes

(3580) Clostridium perfringens alpha toxin were printed on Agilent slides. The small subunit rRNA gene was amplified from fecal DNA using the primers T7prom-Bact-27-for (5′-TGA ATT GTA ATA C GA CTC ACT ATA GGG GTT TGA TCC TGG CTC AG–3′) and Uni-1492-rev (5′-CGG CTA CCT TGT TAC GAC-3′). Samples were initially denatured at 94°C for 2 min followed by 35 cycles of 94°C (30 s), 52°C (40 s), 72°C (90 s), and a final extension at 72°C for 7 min. The PCR products were purified by using the DNA Clean and Concentrator kit (Zymo Research, Orange, NJ, USA). In vitro transcription was performed at room temperature for 2 h with the Riboprobe System (Promega, La Jolla, USA), 500 ng of the T7–16S rRNA gene amplicon, including a 1:1 mix of rUTP and aminoallyl-rUTP (Ambion Inc.