However, as all our study subjects were Caucasians from Finland, genetic variation being thus small between the subjects, the extrapolation of the results to international context would require additional samples from genetically and nutritionally differing areas. As our study provides a link between the host genetic factors and the clustering of the intestinal microbiota in this Finnish cohort, it also warrants further investigations with high-throughput techniques of microbiota analysis to evaluate whether the specific species/OTUs responsible for the microbiota differences can be found, thus potentially enabling

new applications in the field of personalized nutrition and medicine. Methods Subjects and samples P-gp inhibitor One faecal and one blood sample was collected from 79 healthy Caucasian

donors from Southern Finland for the analysis. Pregnant subjects and subjects with diagnosed GIT disorders, regular GIT complaints or antibiotic medication within two months AZD6738 in vitro prior to the faecal sampling were excluded from the study. All subjects were eating mixed diets and subjects on vegetarian diets were excluded. The nutritional intake was not controlled, except for not allowing drastic dietary changes or the habitual use of Selleckchem BIBW2992 probiotic supplements/probiotic-supplemented food products and alcohol prior to the faecal sampling. Body mass index of the subjects was not calculated. The study was approved by the ethical committee of the Helsinki University Hospital and all subjects signed a written informed consent. Faecal samples were collected in containers with anaerobic atmosphere generators, samples were homogenized by mixing and distributed

to 1 g aliquots in an anaerobic cabinet and aliquots were frozen at −70 °C within 5 hour from defecation. The fecal aliquots were processed as described in [26] to isolate the bacterial genomic DNA. Briefly, 1 g of feces was washed to separate the eukaryotic cells from the microbial cells. The collected bacterial mass was pelleted with high speed centrifugation, the pellet was suspended to freeze-thaw buffer and the solution was frozen to −70 °C. A sample for flow cytometry was drawn at this stage. The sample for DNA extraction went through five freeze-thaw-cycles, after which enzymatic (lysozyme, Anacetrapib proteinase K), chemical (sodium dodecyl sulphate) and bead beating techniques were utilized to break down the cells and chloroform-isoamylalcohol-extraction to isolate the bacterial genomic DNA from cell debris. The bacterial genomic DNA was purified using an isolation kit (Blood & Cell Culture DNA Midi Kit Cat no. 13343; Qiagen Inc., USA) according to manufacturer’s instructions. The isolated DNA was diluted to TE-buffer and the DNA concentration was determined using NanoDrop (Thermo-Fisher Scientific, USA). Quality of the DNA was assessed by measuring the ratio 260/280 nm, samples having ratio between 1.7-2.0 and total concentration higher than 20 μg/g were accepted.