Uneven distribution of sequences originating Nutlin-3a mechanism from the different sample types within an OTU was used as criteria for qPCR target selection. Potential primer target sites for specific quantitative analyses were assessed manually from ClustalW 1.83 alignments. Primer 3 online interface[37] and mfold 3.3 DNA-folding servers[38] were used for optimizing the final primer sequences and secondary structure analyses. The primer specificity against publicly available prokaryotic 16S rRNA sequences was checked with FASTA[39] provided by the European Bioinformatics Institute (http://www.ebi.ac.uk/) and against in-house 16S rRNA clone library sequences of human faecal origin, using the blastall option of Parallel BLAST[40] with Corona hardware (http://corona.csc.
fi) maintained by the Finnish IT Center for Science (CSC – Scientific Computing Ltd., Finland). The qPCR primers were synthesized commercially by Oligomer Oy (Helsinki, Finland). The clone sequences used to generate the standard curve in each qPCR assay were classified using The Ribosomal Database Project II Classifier[41]. The assays were named according to the most similar 16S rRNA gene sequence of a cultured bacterial species with the similarity percentages below 98% indicated. qPCR optimization and conditions For each assay, the optimal annealing temperature and MgCl2 concentration were defined using the iCycler iQ Real-Time Detection System (Bio-Rad, Hercules, CA, USA) associated with the iCycler Optical System Interface software (version 2.3; Bio-Rad).
Actual samples were run as triplicates with optimized reaction conditions using SYBR Green I chemistry and 25 ng (specific phylotype targeting assays) or 0.5 ng (universal 16S rRNA gene assay) of faecal bacterial DNA. For all assays, the samples were run with different sample groups randomly mixed in the individual runs to minimize the effect of technical deviation between runs. Amplified clonal 16S rRNA genes were used as standards, ranging from 102 to 107 gene copies per reaction. The reaction mixtures consisted of a 1:75 000 dilution of SYBR Green I (Lonza biosciences, Basel, Switzerland), 10 mmol/L Tris-HCl (pH 8.8), 50 mmol/L KCl, 0.1 % Triton X-100, 2-5 mmol/L MgCl2, 100 ��mol/L each dNTP, 0.5 ��mol/L each primer, 0.024 U Dynazyme II polymerase (Finnzymes, Espoo, Finland) and 5 ��L of either template or water.
The amplification involved one cycle at 95��C for 5 min for initial denaturation, followed by 40 cycles of denaturation at 95��C for 20 s, primer annealing at the defined optimal temperatures for 20 s, extension at 72��C for 30 s and a fluorescence detection step at 80-89��C for 30 s. The specificity of the qPCR assays was checked with a Drug_discovery reassociation curve analysis after amplification by slow cooling from 95��C to 60��C, with fluorescence collection at 0.3��C intervals for 10 s at each decrement.