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BIOgene, USA), which provided a faster, easily scalable FKBPL and equally efficient method compared to the previously described precipitation with trichloroacetic acid and ethanol and subsequent filtration through GF/C filters.35 Purified DNA was dissolved in 200?��l of water and transferred to a scintillation vial with 2?ml of scintillation solution (EcoLume, ICN, USA). Incorporation of the radioactive methyl group was measured using a Beckman liquid scintillation counter. To control the quantity and quality of the DNA, a parallel assay was performed using the dam methylase, which methylates the deoxyadenosine within the sequence GATC (virtually never methylated in humans). Conditions were identical to the M.SssI reaction, except that 6 U of dam methylase were used in 10?mM EDTA, 50?mM Tris-HCl, 5?mM 2-mercaptoethanol buffer. The percentage of methylated CpG sites Selleckchem Trametinib (mCpG%) was estimated from the M.SssI and dam methylase 3H incorporation values as follows: mCpG%=(1�C0.2323 �� M.SssI/dam) �� 100, were 0.2323 is the proportion between GATC sites and CpG sites in the human genome (GRh37). All experiments were performed in triplicate. The standard error of the technique was 3.1��1.8%, CI 95%=[0.4�C5.9%]. No statistically significant difference in CpG methylation level between antrum and corpus was found (mean difference =0.5%, P=0.24, paired t Student test). Therefore, for some of the analyses presented in this paper, methylcytosine content of antrum and corpus was averaged per patient. For comparison with previously published work conducted with different techniques that report the relative methyl-cytosine content (mC%=methyl-cytosine divided by total cytosine content), DNA Damage inhibitor the values were transformed as follows: mCpG%=mC% x (number of cytosines/cytosines in CpG sites in the human genome)=mC% �� 20.7506. Statistics Statistical analyses were performed with R environment for statistical computing.36 Methylation in non-tumor vs. tumor tissue was compared by Student's t-test for paired samples. Differences between two groups were analyzed by Student's t-test for unpaired samples, or by repeated measures ANOVA (rANOVA) when two independent biopsies (antrum and corpus) were available per individual. For comparison of three or more groups, we performed ANOVA with Tukey's Honest Significant Differences method (Tukey's HSD), or one-tailed Cochran-Armitage test for ordinal categorical variables.37 The threshold for abnormal global hypomethylation (AGH) was calculated as the mean percentage of global methylation in controls minus two times the standard deviation. The threshold for enhanced somatic hypomethylation (ESH) was calculated as the mean mCpG% difference between the tumor and the combined value of mucosa, muscularis and serosa, minus two times the standard deviation. Unless otherwise specified, P-values were two-sided. Statistical significance threshold was set at P
