Fig. 3

Sensitive and reliable methodology. A Performance of bisulfite-PCR pyrosequencing and MSRE-qPCR in detecting selected seven DNA methylation standard samples in the TAGMe genomic locus. The x-axis depicts the DNA methylation level; the y-axis in the left depicts the methylation level detected by bisulfite-PCR pyrosequencing, and the y-axis in the right depicts the ΔCt detected by MSRE-qPCR (ΔCt value reflects the DNA methylation level, and the higher value of ΔCt corresponds to the lower methylation level). The repeats of pyrosequencing and MSRE-qPCR were two and three for each grad, respectively. B LOD determination of TAGMe by bisulfite-PCR pyrosequencing. C Determination of the LOD by MSRE-qPCR. The mean ± SD values were plotted. D Heatmap summarizing urine test associations among the first morning (T1-7:00) and three random time (T2-12:00, T3-17:00, T4-22:00) samples in 15 enrolled patients. Pearson correlation coefficients were labeled. P values were calculated using the two-tailed unpaired Student’s t-test. *P < 0.05; **P < 0.01; ns, not significant