Correlation between percentage of methylated reference and T50. Unmethylated reference (bisulfite modified normal donor lymphocyte DNA) and methylated reference (bisulfite modified CpG methylase treated normal donor lymphocyte DNA) were mixed so that the final percentage of methylated reference in the unmethylated reference was 0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The mixtures were amplified using the TIMP3 primers and melted. A) Graph of normalised fluorescence plotted against temperature. Data shown are the mean ± standard deviation of triplicate reactions. B) Linear correlation between percentage of methylated reference and T50.