EZ DNA Methylation-Gold Kit

Protocol tips

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	For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.

Protocol tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.

Publication protocol

DNA from all cell lines was subjected to bisulfite conversion with either the EpiTect Bisulfite Kit (Qiagen), or the EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturers’ protocols. The MJ Mini Personal Thermal Cycler (Bio-Rad Laboratories) was used to perform the conversion reactions. For samples converted with the Qiagen kit, purification and elution was done automatically in the QIAcube System (Qiagen). DNA concentrations were measured both before and after bisulfite conversion using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific).

The promoter methylation status of the six genes CNRIP1, MGMT, SEPT9, SFRP1, SPG20 and VIM was analyzed by qMSP in accordance with the MIQE-guidelines. Primers were purchased from BioNordika Bergman and probes from Life Technologies (see Supplementary Table S1 for sequence information). The EpMotion 5075 pipetting robot (Eppendorf) was used for pipetting samples and mastermix. All samples were run in triplicates in 384-well plates. The total reaction volume per well was 20 μl, including 0.9 μM of each primer, 0.2 μM probe, 1xTaqMan® Universal Master Mix II – no UNG (Thermo Fisher) and bisulfite treated DNA template. The six assays were analyzed in singleplex reactions. In addition to the cell line DNA, each plate contained two methylation-positive controls (commercially available CpGenome Universal Methylated DNA; IVD; Merck Millipore), a non-template control (water), a methylation-negative control (bisulfite treated DNA from healthy donors) and a standard curve consisting of five-fold dilutions of IVD (32.5–0.052 ng). The median quantity (of triplicates) of each of the two methylation-positive controls were averaged and used in calculations of the percent of methylated reference (PMR) values (see below). The qMSPs were run on a 7900HT Fast Real-Time PCR System (Life Technologies) and the amplification involved an initial 10 min at 95 °C, followed by 45 cycles of 15 sec at 95 °C and 1 min at 60 °C. The 7900HT Sequence Detection System version 2.3 (SDS2.3; Life Technologies) was used to analyze the results and the median of quantities was used for calculations. Samples with Cq-value ≥ 35.0 were censored according to the manufacturer’s recommendations. The percentage of methylated molecules per sample (PMR value) was calculated by dividing the normalized quantity (investigated assay/reference) of the samples by the normalized quantity of the methylation positive controls and multiplying by 100 14. ALU was used as a default reference for normalization.

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Manufacturer protocol

Download the product protocol from Zymo Research for EZ DNA Methylation-Gold Kit below.

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