Fig. 3

Real-time PCR amplification plot at cg01009664 of TRH in oral samples. a Example of graph from oral rinse sample of normal control showed only unmethylated product. b Example of graph from oral rinse sample of OSCC patient showed methylated and unmethylated product. c Ten-fold serial dilution for standard curve setting using universal methylated DNA (concentration 10, 1, 0.1, 0.01 and 0.001 ng/μL). d The standard curve of TRH methylation level for methylated DNA calculation. This method calculated methylated DNA under the formula; y = − 3.9127× + 45.926, y was cycle threshold (Ct) whereas x was methylated DNA concentration (ng/μL)