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. Author manuscript; available in PMC: 2014 Sep 18.
Published in final edited form as: Neuron. 2013 Sep 18;79(6):10.1016/j.neuron.2013.08.032. doi: 10.1016/j.neuron.2013.08.032

Figure 2. Measurement of global 5mC and 5hmC levels in the hippocampus following neuronal activation.

Figure 2

(A) LC-MS/MS-MRM chromatograms of nucleosides using three commercial 948-bp standard DNA fragments (dmC .01, dhmC .001, and dC 1.0) showing peaks corresponding to the response obtained from gas phase transitions of dC to C, dmC to mC, and dhmC to hmC. cps, counts per second. (B) Standard curves for 5mC and 5hmC. The percentages of 5mC and 5hmC are plotted against the known ratios of methylated and hydroxymethylated DNA to the total amount of cytosine in the standard samples. (C, D) Validation of HPLC/MS system for 5mC and 5hmC detection accuracy was performed using a set of previously characterized genomic DNA samples (Zymo Research). (E, F) Percentages of 5mC and 5hmC in genomic DNA from several different tissue sources (n = 3/group). (G, H) 5mC and 5hmC levels in area CA1 of adult mice at several time points following flurothyl induced seizures compared to controls (F4, 29 = 13.41; each sample represents the average of 3 technical replicates, n = 6/group). Naive vs. 3 or 24 h. **p < 0.01, ***p < 0.001; one-way ANOVA followed by Bonferroni post hoc test. In figures E- H data are presented as mean ± s.e.m.