Amplified PCR fragments were separated in 8% DGGE gel with denaturing gradient ranging from 45% to 60%. DGGE gels Rabusertib ic50 were run at 70 V for 960 min in a gradient optimised for

each bacterial group (UNIV 38-60%, EREC 40-58%, CLEPT 30-53%, BFRA 30-45%, BIF 45-60% and LACT 38-55%). DGGE gels were stained with SYBRSafe for 30 mins and documented with SafeImager Bluelight table (Invitrogen) and AlphaImager HP (Kodak) imaging system. Digitalised DGGE gel images were imported to the Bionumerics-program version 5.0 (Applied Maths) for normalisation and band detection. The bands were normalised in relation to a marker sample specific for the said bacterial groups. Band search and band matching were performed as implemented in the Bionumerics. Bands and band matching were manually checked and corrected. The principal component analysis was calculated in the Bionumerics.

The PCR-DGGE band intensity data was analyzed with Redundancy Analysis (RDA) [32] using ABO blood group status or presence of B-antigen as grouping factors followed by ANOVA-like Y 27632 permutation test. Bifidobacteria-specific qPCR The qPCR method was applied to detect and quantify the 16 S rRNA gene copies of bacteria, bifidobacteria and four ML323 bifidobacterial species/groups, B. bifidum B. longum group, B. catenulatum/pseudocatenulatum and B. adolescentis in faecal samples [8]. In short, reaction mixture was composed

of 0.3 μM of each primer, PCR Master Mix and faecal DNA diluted 1 ng/μl for bifidobacteria group/species-specific primer pairs and 0.1 ng/μl for universal primers and bifidobacteria stiripentol primers. All the samples and standards were analyzed in three replicates. The results were compared to standard curves for each bacterial group of known concentrations of the bacterial genomic DNA (from 10 ng/μl to 0.0001 ng/μl) and calculated as copies/g wet feces and the detection threshold was set to 107 copies/g. The amplification efficiencies were from 93% to 98% for all the other qPCR primer pairs except for B. bifidum specific primers, in which amplification efficiency varied from 80% to 92% and for B. catenulatum/pseudocatenulatum, in which efficiency varied from 87% to 91%. Acknowledgements P. Salmelainen, S. Lehmonen and the technicians responsible for the blood group determinations are thanked for technical assistance. The volunteers are thanked for the sample donations. References 1. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, Gill SR, Nelson KE, Relman DA: Diversity of the human intestinal microbial flora. Science 2005,308(5728):1635–1638.PubMedCrossRef 2.