Representative positive and negative ZNF582 methylation detection by real-time PCR. For each specimen, ZNF582
m (labeled with FAM) and COL2A (labeled with VIC) were detected simultaneously. Fluorescence data were collected during the annealing/extension step for determination of the crossing point (Cp). The amplification curve for ZNF582 is shown in panel (A) and the control COL2A gene is shown in panel (B). The blue arrow indicates the crossing point, and the x-axis value is the so-called Cp value. The ΔCp is the difference between Cp values for ZNF582
m and COL2A. A smaller ΔCp indicates a higher degree of methylation detected in the testing sample. ZNF582 was deemed to be hypermethylated (or positive) if the ΔCp was smaller than 13 (the cut-off value). The blue arrow is Cp value of ZNF582
m and the green arrow is the un-methylated ZNF582
m in panel (A). ZNF582, zinc finger protein 582; ZNF582
m, methylated ZNF582.