0, 2 mM sodium EDTA, 1.2% Triton® X-100, lysozyme to 20 mg/ml), and incubated

for 30 minutes at 37°C. Next, 25 μL of proteinase K solution and 200 μL of buffer AL were added, followed by an incubation step at 56°C for 30 minutes. DNA concentration was determined using an Eppendorf biophotometer at 260 nm. We obtained similar DNA concentrations after kit extraction both from celiac patients and controls biopsies. A Mann-Whitney U test was performed on total DNA concentration (P = 0.11), indicating a similar amount of extracted DNA in both celiac and controls. PCR amplification Polymerase chain reaction (PCR) was performed, as previously described [17] using 400 VE-822 ng of metagenomic DNA, with minor modification. Briefly, to rule out unspecific PCR products we performed touchdown PCR with a starting annealing temperature of 58°C and decreasing it by 0.5°C each cycle to reach 53°C, then 30 cycles at 53°C were achieved. Same amounts of amplified DNA were also obtained. A Mann-Whitney U test was performed on PCR amplicons (P = 0.23), indicating a similar amount of PCR products in BMN 673 solubility dmso both celiac and controls. To minimize heteroduplex formation and single-stranded

DNA (ssDNA) contamination during PCR amplification that might cause sequence heterogeneity in a single TTGE band, an additional 5 cycles of reconditioning PCR was performed, taking 1/10 of the previous PCR volume as template in a new reaction. Moreover, we used 16S rDNA V6-V8 region instead of V3-V4 region that showed coamplification with human DNA. To avoid the problem due to the low bacterial load we performed six individual PCR reactions for each sample. The individual PCR reactions were unified,

analyzed by electrophoresis on 2% agarose gels containing ethidium bromide to determine their size (498 bp), and concentrated with SpeedVack (Savant, SN-38 supplier Holbrook, NY, USA). The unified PCR reactions, before and after the concentration step, were titrated using two different methods: first, densitometry analysis of agarose gel by GelQuest software (Sequentix, Klein Raden, Germany); second, measure of DNA density by biophotometer at 260 nm. The results obtained by such measures were in agreement GPX6 each other. PCR protocol was optimized to obtain maximum yield from starting total DNA. The band intensity was quantified at every step (touchdown PCR, reconditioning PCR, concentrated PCR) to ensure an equal DNA concentration. A first-step assessment of DNA suitability for subsequent PCR was achieved through a β-globin gene amplification for each starting sample. Briefly, aliquots of each DNA sample (50 ng) were amplified with specific primers: forward primer, 5′-CAACTTCATCCACGTTCACC-3; reverse primer, 5′-GAAGAGCCAAGGACAGGTAC-3′.