Universal methylated human dna standard

DNA was isolated from skin samples using High Pure PCR template (Roche). Bisulfite conversion was performed with EZ DNA Methylation kit (Zymo Research) following the manufacturer’s instructions using 350 ng of DNA. After conversion, DNA was eluted in 12 μl. For positive controls and standard curve of methylated primers Bisulfite Converted Universal Methylated human DNA Standard (Zymo Research) was used. Standard curve of unmethylated primers was performed using DNA from a control subject. As control of conversion reaction, we used Universal Methylated human DNA Standard (Zymo Research). Primers used for methylation specific PCR were next: UCHL1 Forward 5′-TCG TAT TTA TTT GGT CGC GATC-3′, Reverse 5′-CTA TAA AAC GCC GAC CAA ACG-3′, unmethylated UCHL1 forward 5′-GGT TTG TAT TTA TTT GGT TGT GAT T-3′, Reverse 5′-CAA CTA TAA AAC ACC AAC CAA ACA-3′^{17 (link)}. PCR was performed in 10 μl final volume using 25 ng of converted DNA, 0.3 mM each primer and 5 μl of GoTaq qPCR Master mix (Promega). To correct for DNA input, we used a primer pair specific for a sequence of beta-actin where no CpG sites are present and described previously. ActB Forward 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′, ActB Reverse 5’-AACCAATAAAACCTACTCCTCCCTTAA-3′^{18 (link)}. Data were represented as methylation ratio: $$\text{Methylation}\text{ratio}=\frac{\text{UCHL1}\text{methylated}}{\text{Beta-actin}}/\frac{\text{UCHL1}\text{unmethylated}}{\text{Beta-actin}}$$

Up to 0.5 μg of plasma cfDNA and 1 μg of gDNA and were chemically modified with sodium bisulfite (SB), in order to convert only the non-methylated cytosines to uracils and not the methylated ones. SB conversion was performed with the EZ DNA Methylation-Gold^TM Kit 200 (ZYMO Research, Irvine, CA, USA), according to the manufacturer’s instructions, as previously described [35 (link)]. The Universal Methylated Human DNA Standard (ZYMO Research, Irvine, CA, USA) was used as the 100% methylated control. In each conversion reaction, the dH₂O and gDNA from the 100% methylated control were used as negative and positive controls, respectively. To evaluate the quality of the SB converted DNA in our samples, we applied a specific MSP assay for ACTB, as previously described [34 (link)]. Real-time PCR amplification occurred in all of the SB converted DNA samples. The SB converted DNA was stored at −70 °C until use.