Comparison between MSRE-qPCR and MS-qPCR

Six standard DNA samples (800 ng each) were generated by combining different proportions of untreated and artificially methylated blood DNA (produced using M.SssI CpG Methylase; Zymo Research, E2011). Specifically, samples 1 to 6 contained 100%, 33.3%, 11.1%, 3.7%, 1.23%, and 0.41%, of artificially methylated DNA. In total, 400 ng of each sample was bisulfite-treated using EZ DNA Methylation-Lightning Kit (Zymo Research, D5031), while the remaining 400 ng was digested using 10U of the methylation-sensitive restriction enzyme HpaII (New England BioLabs, R0171S) and purified with DNA Clean and Concentrator-5 (Zymo Research, D4013). Samples were then amplified by qPCR (CFX96 Touch qPCR System, Bio-Rad) using primers designed for bisulfite-converted methylated DNA (MS-qPCR) and genomic untreated DNA (MSRE-qPCR). Primers (sequences available upon request) have similar efficiencies and were designed on the same region of the CpG island of the human MGMT gene, which is known to be unmethylated in blood samples collected from healthy donors (Additional file 2: Table S1). Both amplicons have similar length and were amplified in triplicates in 20 µl of reaction containing ZymoTaq qPCR Premix (Zymo Research, E2055), 0.4 µM of each primer, and the equivalent of 100 ng of input DNA prior to bisulfite conversion or digestion. The program used to generate both amplicons has an initial 10-min denaturation step at 95 °C followed by 40 cycles of denaturation at 97 °C for 20 s and annealing/extension at 58 °C for one minute. Data were analyzed using Bio-Rad CFX Maestro Software (Bio-Rad).

Methylation of the human MGMT target region in untreated and artificially methylated blood DNA was determined by MSRE-qPCR according to the protocol described above. Results are represented in Additional file 2: Table S1.