Cpgenome universal methylated and unmethylated dna

Genomic DNA was extracted using a QIAamp DNA Mini Kit (QIAGEN, USA). Genomic DNA was treated with sodium bisulfite, and the methylation status of the promoter region SQC, Squamous cell carcinoma: ADC, adenocarcinoma of the TGFBI gene was analysed using MSP with primers specific to either unmethylated or methylated alleles. Primers used for the methylated reaction were 5′-GCCGCCCGCTTG CCCGTCGGTC-3′ (sense) and 5′-AAACCCGCCAAAATCGC GACG-3′ (antisense) primers. Primers used for the unmethylated reaction were 5′-GCTGCCTGCTTGCCTGTTGGTT-3′ (sense) and 5′-AAACCCACCAAAATCACAACA-3′ (antisense). All polymerase chain reaction (PCR) were carried out using reagents supplied in the GeneAmp DNA Amplification Kit using AmpliTaq Gold as the polymerase (PE Applied Biosystems, USA) on a PTC-100 instrument (MJ Research, Watertown, MA, USA). CpGenome™ Universal methylated and unmethylated DNA (Chemicon, USA) was used as the positive controls for methylated and unmethylated genes, respectively. Negative control samples without added DNA were included for each PCR run. PCR products were analysed on a 2% agarose gel, stained with ethidium bromide, and visualized under UV light. Each MSP run was repeated at least once to confirm the results.

Genomic DNA was extracted from cultured cells and primary tumor tissues by the QIAamp DNA Mini kit (Qiagen, Valencia, CA, USA) and subjected to bisulfite treatment with the EZ DNA Methylation-Gold kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s instruction. Bisulfite-modified DNA was amplified with PCR primers, and the purified products were assayed on the PyroMark Q96MD System (Qiagen), as previously described (Kim et al. 2018 (link)). The bisulfite PCR primer and sequencing primer sequences are shown in Supplementary Table 1. The degree of methylation was expressed for each DNA locus as the percentage of methylated cytosines among all cytosine residues (methylated and unmethylated), and the methylation level was calculated from the mean methylation percentage of the six CpG sites evaluated. Non-CpG cytosine residues were used as built-in controls to verify bisulfite conversion. Three controls were included for each pyrosequencing run. One well was filled with water to ensure no contamination, and two wells were filled with CpGenome universal methylated and unmethylated DNA (Chemicon, Temecula, CA, USA) as positive and negative controls, respectively, to assess the repeatability of the assay.

Alu methylation analysis was quantitatively performed on the bisulfite-treated DNA using pyrosequencing with the polymerase chain reaction (PCR) primers and conditions previously described (Bollati et al., 2007 (link)). In brief, the bisulfite-treated samples (50 ng) were amplified with a biotin-labeled primer via PCR, which enables the conversion of the PCR product to a single-stranded DNA template suitable for pyrosequencing. Confirmation of the quality of the PCR products and their freedom from contamination was established on 2% agarose gels with ethidium bromide staining. After purification of PCR products using Sepharose beads on PyroMark Vacuum Prep Workstation (Qiagen), pyrosequencing was carried out using the PyroMark Q96MD System (Qiagen) according to manufacturer’s instructions, including a single-strand binding protein. The percentage of methylatation was expressed for each DNA locus as % 5-mC divided by the sum of methylated and unmethylated cytosines. We tested each marker 3 times and used the average percentage in the statistical analysis. Three controls were included in every pyrosequencing run. One well was filled with water to ensure no contamination, and 2 wells were filled with CpGenome Universal methylated and unmethylated DNA (Chemicon, USA) to evaluate the repeatability of the assay.