The PCR was carried out under the following conditions: 1 cycle of 95°C for 7 min, 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min and 1 cycle of 72°C for 7 min. 500 ng of DNA of PCR product from each sample were used to perform the subsequent TTGE experiments. TTGE analysis of PCR amplicons We used the DCode Universal mutation detection system (Bio-Rad, Paris, France) for the sequence-specific separation PF-6463922 purchase of PCR products. Electrophoresis was performed as previously described . TTGE runs were conducted in triplicate and gel photographed with DigiDoc-It system (UVP, Cambridge, UK). Species-specific PCR We choose to detect those particular species whose presence seems
to be involved in celiac disease [7, 9]. 16S rDNA gene-targeted primers were utilized to detect them. The primers used were ECO-1 5′-gacctcggtttagttcacaga-3′, ECO-2 5′-cacacgctgacgctgacca-3′ for Escherichia coli (585 bp); BV-1 5′-gcatcatgagtccgcatgttc-3′, BV-2 5′-tccatacccgactttattcctt-3′ for
Bacteroides vulgatus (287 bp); g-Ccoc-F 5′-aaatgacggtacctgactaa-3′, g-Ccoc-R 5′-ctttgagtttcattcttgcgaa-3′ MK-4827 for Clostridium coccoides group (438-441 bp), g-Bifid-F 5′-ctcctggaaacgggtgg-3′, g-bifid-R 5′-ggtgttcttcccgatatctaca-3′ for Bifidobacterium spp (549-563 bp). The PCR were performed as previously described . Data Analysis Agglomerative Hierarchical Classification (AHC.) Dendrogram generated with XLStat 7.5 (Addinsoft, NY, USA) on binary matrix of TTGE variables was evaluated by one-tailed chi-squared test. Data were automatically mean centred and unit variance (UV) scaled. A P value equal or less 0.05 was considered statistically significant. Dice similarity index (S D , mean % ± SD) was calculated within the
respective HC and CD groups to assess inter-individual similarity by the formula S D = (2n AB )/(nA + nB), where n A is the total clonidine number of bands in pattern A, n B is the total number of bands in pattern B and n AB is the number of bands common to pattern A and B. Ecological features. Doc-It LS software (UVP, Cambridge, UK) was used for TTGE bands densitometry peak height quantification, and the correspondent data were analyzed for the microbial Protein Tyrosine Kinase inhibitor biodiversity by Shannon-Wiener index with SigmaPlot 9.0 software. Intra-group variance value (V value) was also calculated. V value defines the variance of data points in each cohort, representing the data dispersion, and indicating the homogeneity/heterogeneity between individuals within a population. In addition, the range-weighted richness (Rr), reflecting the carrying capacity of the duodenal system, was calculated by the formula Rr = N2 XTg, where N is the total number of bands in the TTGE profile and Tg the temperature gradient comprised between the first and the last band of the same pattern . Principal Component Analysis (PCA). Linearly-dependent TTGE variables were ortogonalized in new factorial axes (F1,F2…Fn) through PCA by XLStat 7.5 (Addinsoft).