, 2009). In short, the SSU rRNA gene was amplified from 10-ng DNA with the T7prom-Bact-27-for selleck chemicals Pazopanib and Uni-1492-rev primers followed by in vitro transcription and labeling with Cy3 and Cy5, respectively. The Cy3/Cy5-labeled target mixes were fragmented and hybridized on the arrays at 62.5��C for 16h in a rotation oven (Agilent Technologies, Amstelveen, The Netherlands). After washing and drying, the slides were scanned. Data was extracted from the microarray images using the Agilent Feature Extraction software, versions 7.5�C9.1 (http://www.agilent.com) and subsequently, the microarray data normalization, and further analyzed using a set of R-based scripts (http://r-project.org) in combination with a custom-designed relational database that runs under the MySQL database management system (http://www.

mysql.com;Rajili?-Stojanovi? et al., 2009). Hierarchical clustering of probe profiles was carried out using the Euclidian distance and Ward’s minimum variance method. These small intestinal microbiota composition profiles generated for the samples obtained from healthy subjects were compared with the ileostoma effluent profiles that were already present in the database (Booijink et al., 2010). Multivariate statistical software Canoco 4.5 for windows (Leps and Smilauer, 2003;Biometrix, Plant Research International, Wageningen, The Netherlands) was used to perform the principle component analysis on log-transformed HITChip probe signal-intensity profiles. High molecular weight DNA isolation from ileostoma effluent samples From the ileostoma effluent samples, 30�C40g were used for high molecular weight DNA isolation as described by Berridge et al.

(1998) with modifications. Effluent samples were resuspended as portions of 10g in 20ml 1 �� phosphate-buffered saline containing 10�C20 glassbeads of 2mm and vortexed for 3min. After centrifugation at 700g for 1min to remove large-sized debris, the supernatant was centrifuged at 3000g for 10min. The pellet was resuspended in 5ml 1 �� phosphate-buffered saline with 2% w/v polyvinylpyrrolidone (PVP), incubated at room temperature for 5min and centrifuged at 3000g for 10min. After washing with 1 �� phosphate-buffered saline, the pellet was resuspended in 3ml lysis buffer (50mM glucose, 50mM Tris-HCl (pH 8.0), 100mM EDTA, 2% w/v PVP, 150mM NaCl, 0.2mgml?1 RNAse A (Sigma-Aldrich, Zwijndrecht, The Netherlands), 0.

02mgml?1 mutanolysin (Sigma-Aldrich) and 1mgml?1 lysozyme (Sigma-Aldrich)), and incubated at 37��C Brefeldin_A for 2h. Following addition of 360��l 10% w/v SDS and 160��l of a 10mgml?1 Proteinase K (Sigma-Aldrich) solution, the samples were incubated at 45��C for 2h. After addition of 600��l 5M NaCl and 480��l CTAB/NaCl solution (10% Cetyl trimethylammonium bromide in 0.7M NaCl), samples were incubated at 65��C for 10min.