Epitect plus bisulfite kit

DNA methylation levels in CpG islands were analyzed using the Quantitative methylation‐specific PCR (qMSP) assay. Genomic DNA was extracted from paraffin sections using the Epitect Plus FFPE Lysis kit (Qiagen, Venlo, Netherland). The DNA samples were treated with sodium bisulfite using the EpiTect Plus Bisulfite Kits (Qiagen). DNA methylation of E‐cadherin and p16^ink4a genes were analyzed by SYBR green‐based qMSP. The methylated and unmethylated primers were designed as described in a previous study (Table 1) (Herman et al., 1996). PCR was performed in a total volume of 25 μl, with 1.0 μl of bisulfite‐treated DNA template mixed with 12.5 μl of KAPA SYBR FAST qPCR Kit (Nippon Genetics, Tokyo, Japan) and a pair of primers at a final concentration of 400 nM. The PCR conditions included initial incubation at 50°C for 2 min, denaturation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, and annealing at 58°C for 1 min. After PCR amplification, a dissociation curve was generated to confirm the size of the PCR product. The percentages of E‐cadherin and p16^ink4a methylation in a sample were estimated using the following formula (Lu et al., 2007): Methylation (%) = MM+U×100=11+UM×100=11+2−ΔCq×100, where M and U are the copy numbers of methylated and unmethylated E‐cadherin/ p16^ink4a, respectively, and ΔCq = Cq_U − Cq_M.

Genomic DNA was extracted from the buccal smear samples using Gentra® Puregene® buccal cell kits (Qiagen), following the manufacturer’s instructions. The extracted DNA samples were treated with sodium bisulfite using the EpiTect® Plus Bisulfite Kits (Qiagen), and DNA methylation of the SIRT1 gene was performed using the methods described previously [38 (link)]. Each experiment was performed in triplicate. Data are expressed as mean ± SD of the DNA methylation.

Genomic DNA was isolated by a PureLink™ Genomic DNA kit (Invitrogen). For pyrosequencing, 1 µg was subjected to bisulfite-conversion using an EZ DNA methylation kit (Zymo Research, Orange, CA). All primers for methylation experiments and other assays (qRT-PCR, construction of plasmids, etc) are listed in Supplementary Data File 9.
For EpiTPYER assays, 2 µg of genomic DNA was bisulfite treated using an EpiTect Plus Bisulfite Kit according to the manufacturer’s instruction (Qiagen, Leipzig, Germany). PCR primers were designed using EpiDesigner (Sequenom, http://www.epidesigner.com). PCR condition for EpiTPYER was: 15 min at 94 °C (for initial denaturation); 45 cycles of 94 °C for 20 s, 56–62 °C for 30 s, 72 °C for 60 s (for amplification); 72 °C for 3 min (for final elongation). EpiTPYER was performed with EpiTYPER Reagent and SpectroCHIP Set (Agena Bioscience, San Diego, CA) according to the manufacturer’s instruction, data were analyzed using EpiTYPER^TM ver 1.2 (Agena Bioscience), and a heat map was drawn by the gplots package (R 2.9.1).

For methylation analysis of the IG-DMR and the Meg3-DMR, total cellular DNA was extracted using DNeasy Tissue kit (Qiagen) according to manufacturer’s protocol. One μg of DNA was bisulfite treated using EpiTect Plus Bisulfite kit (Qiagen). DNA fragments covering the IG-DMR and the Meg3-DMR, respectively, were amplified by PCR using the following primers: IG-DMR-F: 5′-TGGGATTATAGGTATTATGTTTGGA-3′, IG-DMR-R: 5′-CACTACTAAAAACTACATTTAAACAA-3′, Meg3DMR-F 5′- GTTAGGGATTAATTTTTATGTGTTAG-3′, Meg3DMR-R 5′-CAAATTCTATAACAAATTACTCTAAC-3′.
The IG-DMR fragment (909 bp) harbors 31 CpG dinucleotides and the Meg3-DMR (819 bp) harbors 44 CpG dinucleotides. According to the sequence NT_026437.12 at NCBI Database the position of the analyzed IG-DMR sequence is 82.276.640 – 82.277.549 and that of the analyzed Meg3-DMR fragment is 82.291.515 – 82.292.333. PCR products were cloned into pCR4-TOPO vector using TOPO TA cloning kit (Life Technologies) and sequenced. Methylation was analyzed using BiQ-Analyzer software
[16 (link)].

Genomic DNA extraction of fresh-frozen tissues was performed using the DNeasy® Blood & Tissue Kit (QIAGEN, Crawley, UK) according to the manufacturer's instructions. Approximately 2 mm3 (link) tissue was used, and purified genomic DNA was made up to a final volume of 200 µl. DNA extracts were quantified and assessed for quality using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Loughborough, UK). Bisulphite modification of 1 µg genomic DNA was performed using the EpiTect® Bisulfite Kit (QIAGEN), according to the manufacturer's instructions.
Paraffin-embedded blocks were sectioned at 5 µm thickness and mounted on glass slides. A reference slide for each sample was stained with haematoxylin and eosin. Areas containing more than 80 per cent of tumour tissue were marked to guide sampling from adjacent, non-haematoxylin and eosin-stained sections. DNA extraction and bisulphite treatment of formalin-fixed, paraffin-embedded tissues was performed using the EpiTect® Plus Bisulfite Kit (QIAGEN), according to the manufacturer's instructions.