Episcope methylated hct116 gdna

EpiScope® Unmethylated HCT116 DKO gDNA and EpiScope® Methylated HCT116 gDNA reference cell-line DNA samples (catalog #3521, #3522 Takara Bio, Moutainview CA) were sheared to approximately 180 bp by acoustic shearing to mimic fragmented cfDNA using a Covaris Ultrasonicator (Covaris, Woburn MA). DNA size distribution of sheared DNA was tested using Agilent 2100 Bioanalyzer High Sensitivity DNA Kit (catalog #5067–4626, Agilent Technologies, Santa Clara CA) and quantified by Qubit dsDNA High Sensitivity kit (catalog #Q32854, ThermoFisher Scientific, Waltham MA). Fully methylated and unmethylated DNA were mixed to obtain the following ratios of methylation: 0, 10, 25, 50, 75, 100%. Standard curves with known methylation ratios were included in the assay to deduce the methylation level of tumor samples.

DNA from peripheral blood leukocytes was used as a control for unmethylated RUNX3, and EpiScope methylated HCT116 gDNA (Takara Bio Inc.) was used as a control for hypermethylated RUNX3. After the serum samples were thawed from −80 °C, DNA was extracted from 0.4 mL of each sample with a MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche, Tokyo, Japan) in accordance with the manufacturer’s instructions. The DNA was eluted in 50 µL of elution buffer, and Qubit 2.0 fluorometers (Thermo Fisher Scientific, Yokohama, Japan) were used to quantify the double-stranded DNA.

Genomic DNA was obtained from tumor and normal mucosal tissues using the QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France). DNA was subjected to bisulfite treatment, as described previously. [32 (link)] The bisulfite-modified DNA was used as a template for fluorescence-based real-time PCR. [35 (link)] The amplifications were performed using the TaKaRa Thermal Cycler Dice™ Real Time System TP800 (TaKaRa, Tokyo, Japan). The Q-MSP primers for methylated DNA were Q-MSP-ACTB-F (5′-TGGTGATGGAGGAGGTTTAGAAGT-3′) and Q-MSP-ACTB-R (5′-AACCAATAAAACCTACTCCTCCCTTAA-3′). A standard curve was generated using serial dilutions of universal methylated DNAs (EpiScope™ Methylated HCT116 gDNA; TaKaRa, Tokyo, Japan). The normalized methylation value (NMV) was defined as follows: NMV = (TRGs-S/TRGs-FM)/(ACTB-S/ACTB-FM), where TRGs-S and TRGs-FM represent TRG methylation levels in the sample and universally methylated DNAs, respectively, and ACTB-S and ACTB-FM correspond to b-actin in the sample and universally methylated DNAs, respectively. To analyze the methylation status of p16 [36 (link)], RASSF1A [36 (link)], CDH1 [36 (link)], CDH13 [37 (link)], MGMT [38 (link)], DAPK [38 (link)], DCC [39 (link)], COL1A2 [25 (link)], TAC1 [30 (link)], SST [40 (link)], and GALR1 [32 (link)], primers and conditions were used as previously described.