For qPCR analysis, total DNA was isolated from 1 g of faecal selleck bio material according to Apajalahti et al, which included removing the undigested particles from the faecal material by three rounds of low-speed (200 �� g) centrifugation and collection of the bacterial cells with high-speed centrifugation (30 000 �� g) at 15��C for 15 min using a Beckman AvantiTM centrifuge (Fullerton, CA, USA) with the rotor JA 25.50 or JLA 16.250 rotor, respectively. The bacterial cells were lysed after centrifugation with a combination of freeze-thaw cycles (freezing for 1 h at -70��C and thawing for 15 min in a 37��C water bath), lysozyme and vortexing with glass beads. DNA concentrations were determined with a NanoDrop ND-1000 Spectrophotometer (NanoDrop products, Wilmington, DE, USA).
Design of qPCR assays Divergences detected by comparing the sequence data of 16S rRNA gene clone libraries of healthy controls and symptomatically sub-grouped IBS patients (diarrhoea-predominant IBS, IBS-D; constipation-predominant IBS, IBS-C; and mixed symptom-subtype IBS, IBS-M) were used as the basis for selection of qPCR targets. Prior to cloning and sequencing, the faecal microbial genomes had been profiled and fractioned on the basis of genomic guanine-plus-cytosine content. Partial 16S rRNA gene sequences encompassing the variable regions V1 and V2 combined from all four sample types were aligned using either the version Beta 2003-08-22 of ARB[11,32] or ClustalW 1.83. For the ARB alignment, an aligned sequence database (ssu_jan04_corr_opt.arb) was downloaded from the ARB home page (http://www.
arb-home.de) and the in-house sequences were aligned using the ARB-EDIT FastAlign function, followed by manual correction of the alignments with special attention to the ends of the sequences. Finally, the sequences were imported into an existing tree file of the database (Tree-Bacteria) by filtering the data against a sequence of similar length as the imported partial 16S rRNA Drug_discovery gene sequences. Regions of the tree where sequences derived from one subject group (healthy vs IBS or healthy vs IBS subtypes) dominated over the other groups were considered as potentially interesting. In addition, a ClustalW 1.83 alignment (FAST DNA pairwise alignment algorithm option, gap penalty 3, word size 4, number of top diagonals 1 and window size 1) was constructed covering approximately 450 bp from the 5�� end of the 16S rRNA gene and visually inspected and cut from the Escherichia coli position 430 (universally conserved GTAAA) with BioEdit version 22.214.171.124. Distance matrices were calculated from the ClustalW alignment with Phylip 3.66 Dnadist using Jukes-Cantor correction.