Sodium Bisulfite Mapping of DNA Methylation Status Genomic DNA was extracted (DNeasy, Qiagen) and sodium bisulfite conversion of genomic DNA was performed as previously described [55,56] for 13 suicide subjects and 11 controls for hippocampal samples and for 4 suicide subjects and 4 controls for cerebellum samples. Primers for PCR were directed against the rRNA gene promoter using the following sequences: sense: 59-GTT TTT GGG TTG ATT AGA-39; antisense: 59-AAA ACC CAA CCT CTC C-39 [32]. Because the primers did not contain CpG dinucleotides, methylated and unmethylated sequences amplified with equal efficiency. The resulting product was excised, purified, subcloned, and transformed (TA cloning kit, Invitrogen). Individual clones were extracted and sequenced (CEQ 8800, Beckman-Coulter) according to the manufacturer’s protocol. Twenty clones were sequenced for each subject from 2 to 3 independent PCR reactions. To ensure that the bisulfite conversion was complete, only clones in which all cytosine residues in non-CpG dinucleotides had been converted to thymidine were included in the analysis. Nearest Neighbor Quantification of Methylated Cytosine Content Genome-wide levels of 5-methylcytosine were quantified as previously described [57]. Briefly, genomic DNA from the same subjects as those used for bisulfite analysis was subjected to MBoI restriction enzyme digestion (recognition sequence: NGATCN), incubated with a 32P-labelled oligonucleotide, loaded onto TLC phosphocellulose plates, and separated by chromatography. Reactions were repeated in triplicate. The intensities of 5- methylcytosine and cytosine spot densities were analyzed using a PhosphoImager screen followed by Image Quant image analysis. For each subject, levels of methylated cytosine were tabulated as a percentage of total cytosine content, following the formula: [(5- methylcytosine) 6 100]/(5-methylcytosine + cytosine). RNA Extraction and Quantitative Reverse Transcription PCR (qRTPCR) of rRNA Gene Expression RNA extraction was performed using Trizol (Invitrogen) followed by Dnase I treatment, and cDNA conversion was performed using random hexamer primers (Invitrogen) according to the manufacturer’s instructions (Roche Molecular Biochemi- cals). The subjects were the same as those used for the bisulfite mapping study, however, RNA samples from subjects with RNA Integrity Numbers (RINs) lower than 5.0 or brain pH less than 6.0 were excluded from analysis (N = 2 suicide subjects and N = 3 control subjects), consistent with previously described criteria for exclusion [50]. Because of the low number of remaining subjects and preliminary data indicating a trend for lower rRNA expression in the suicide subjects used for bisulfite analysis, we included an additional 5 suicide subjects (total N = 16) and 1 control subject (total N = 9) for rRNA expression analysis. Primers were directed against the rRNA gene using the following sequences: sense; 59-TTC TCT AGC GAT CTG AGA GGC GT-39, antisense; 59-TAC CAT AAC GGA GGC AGA GAC AGA-39 and GAPDH (Genebank accession number NM_002046) using the following sequences: sense; 59-GAA GGT GAA GGT CGG ACT C-39, antisense; 59-GAA GAT GGT GAT GGG ATT TC-39. A master mix, containing the cDNA, 10 mM Tris-Cl, 50 mM KCl, 2.0 mM MgCl2, 0.2 mM dNTPs, 5X SYBR Green I Solution, HotStart Taq DNA polymerase (Superarray Bioscience Corporation), and 0.2 mM of the sense and antisense primers, were loaded into LightCycler capillaries (Roche Molecular Biochemi- cals). For the rRNA gene, the qRTPCR protocol (LightCycler Software 3.5, Roche Molecular Biochemicals) consisted of initial rRNA Methylation in Suicide HotStart Taq DNA polymerase activation cycle (15 min, 95uC, with a temperature transition rate of 20uC/sec) followed by 36 cycles of denaturation (30 sec, 95uC, with a temperature transition rate set at 20uC/sec), annealing (30 sec, 55uC, with a temperature transition rate set at 20uC/sec) and elongation (30 sec, 72uC, with a temperature transition rate set at 20uC/sec). A single fluorescence reading was acquired at the end of each elongation step. Subsequently, the PCR products were melted using the following program: 95uC with a temperature transition rate of 20uC/sec, 65uC, with a temperature transition rate of 20uC/sec followed by 95uC, with a temperature transition rate of 0.1uC/sec. The presence of a single melting peak followed by analysis on 1.5% agarose gel confirmed product specificity. For the GAPDH gene, the above procedure was identical except for annealing temperatures of 51uC and 61uC during the PCR and melting steps, respectively. Reactions were repeated in triplicate. Reactions were also carried out in the absence of reverse transcriptase to verify the absence of genomic DNA contamination. To determine the relative concentrations of rRNA gene expression, a standard curve of 10-fold serial dilutions of a mixture of each of the sample cDNA was used to plot the relative Ct value for each gene on the y-axis and the amount of cDNA used on the x-axis. To calculate the fold-change, the relative amount of rRNA product was divided by the relative amount of GAPDH for each subject. Statistical Analyses Statistical analyses were carried out using Statview (Cary, NC). For DNA methylation analysis, a factorial ANOVA was carried out with the percentage of methylation as the dependent variable and group (suicide subjects and controls) as the between groups factor. The data were then subjected to Bonferroni Post-hoc analysis to examine methylation status between groups across all CpG sites. A standardized effect size and associated 95% confidence interval of the methylation differences between suicide subjects and controls was calculated for each CpG site using the difference between group means divided by a pooled standard deviation corrected for bias, according to previously described methods [58,59]. The analysis of the relationship between DNA methylation at each CpG site between suicide subjects and controls was conducted using linear regression, as were analyses of the relationships between DNA methylation, expression, PMI, brain pH, and age. To identify possible diagnostic variables influencing methylation status, factorial ANOVA followed by Bonferroni Post-hoc comparisons were used to compare groups of subjects with different clinical diagnoses. For nearest neighbor as well as rRNA expression analysis, unpaired t-tests were used to examine differences between the suicide and control groups. Data from these statistical analyses are presented as mean 6 SEM. Statistical significance was determined at P#0.05. Supporting Information Figure S1 The published rRNA promoter sequence U13369 is followed by the sequencing results for each subject for the region examined by sodium bisulfite mapping. The base pair length of each sequence is listed on the right side and the consensus sequence at the bottom of the sequencing results. Found at: doi:10.1371/journal.pone.0002085.s001 (0.38 MB PDF) Acknowledgments We thank Lilian Canetti for histological assistance in obtaining tissue samples for analysis, Pierre Bernard for his help in collating the subjects’ clinical information. PLoS ONE | www.plosone.org 8 May 2008 | Volume 3 | Issue 5 | e2085