The slightly lower Ct-value for the M extraction may be caused by the fact that the DNA concentration was initially higher for this method and thus template DNA was diluted more prior to qPCR analysis. Further analysis of qPCR data showed that in seven of nine cases the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly

higher for fecal samples that had been frozen prior to DNA Deforolimus extraction compared to the fresh samples extracted with the same kit (Fig. 3a). The extent of shift in the Firmicutes to Bacteroidetes ratios between frozen and fresh samples appeared to depend on both extraction method and donor in an unpredictable manner, but was on average 2.2-fold (SEM = 0.52) higher for samples that had been frozen. Analogous comparisons were made for ratios of the total bacteria as determined from two different 16S rRNA gene regions (Eub1 and Eub2) by separate qPCR assays. In this case, no significant difference was observed between the I-BET-762 order frozen and fresh samples extracted with the same kit, and the calculated average change in ratios was indeed 1.0, SEM = 0.03 (Fig. 3b). This observation strengthens the confidence of the previous finding, which may in general suggest relatively better extraction or stability

of PCR amplifiable DNA from gram-positive bacteria (Firmicutes) following freeze storage. This could be caused by differences in the cellular composition of gram-positive and gram-negative bacteria. Random shearing

of DNA during freeze storage is not likely to bias the qPCR-determined ratios of Firmicutes to Bacteroidetes 16S rRNA genes, because the amplification products were identical in length (Table 1). In most cases, both an increase in the overall relative abundance of Firmicutes and a corresponding decrease in relative abundance of Bacteroidetes 16S rRNA genes were observed in connection with freeze storage (Fig. 4). Also in eight of nine cases, a decrease in the relative ratio was also observed for the Bacteroidetes species B. thetaiotaomicron, which is consistent with the findings for the phylum as a whole. For the Enterococcus spp., belonging to the Firmicutes phylum, however, only Branched chain aminotransferase a slight tendency for an increase with freezing was observed, which may be due to the near detection limit overall abundance of this genus (Fig. 4). In conclusion, the data presented in this study indicate that freeze storage of human fecal samples prior to DNA extraction affects downstream qPCR analysis of community composition and thus should be given due consideration during study design. This could be achieved by direct DNA extraction on fecal samples or, for comparisons, by ensuring that all samples have been frozen in a similar manner.