2.4. DNA Methylation Analysis of CpGs by iPlex MassARRAY

Genomic DNA was extracted from peripheral blood cells using the GeneJET kit following the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA, USA). The concentration and purity of the isolated DNA were determined using a Qubit bench fluorimeter (Thermo Fisher Scientific, Waltham, MA, USA) and a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The isolated DNA was then subjected to bisulfite conversion using the EZ DNA Methylation Kit (Zymo Research, Tustin, CA, USA), following the manufacturer’s instructions. Briefly, 500 ng of genomic DNA was treated with bisulfite, and the modified DNA was eluted with 30 μL of water. Methylation analysis was performed, employing MALDI-TOF-MS technol-ogy with the MassARRAY system (iPLEX assay, Agena Bioscience, San Diego, CA, USA) according to the manufacturer’s protocol. The obtained results were analyzed using the MassARRAY Typer Analyzer 4.0 software provided (Agena Bioscience, San Diego, CA, USA.

The size and quality of products after the first PCR were visualized on 1.5% agarose gels with ethidium bromide under ultraviolet (UV) light.

The method for methylation assessment is based on the detection of products generated after the primer extension reaction. Initially, locus-specific PCR is carried out using a pair of primers designed for the region of interest. Subsequently, a second PCR is performed using ddNTPs. Moreover, there is shrimp alkaline phosphatase (SAP) step between the first PCR and second one. SAP catalyzes the removal of phosphate groups from the 5′ ends of dNTPs that enhance the single-nucleotide primer extension reaction [38].

During the second PCR, the extension primer is annealed near the CpG site of interest and is extended by one nucleotide. The elongation products have varying masses depending on the nucleotide incorporated, reflecting the methylation status of the CpG site in the original DNA sample (Figure 2).

The scheme for determining methylation by MALDI-TOF MS. At the initial stage, DNA is isolated from blood cells. The next step is bisulfite conversion, which makes it possible to distinguish between methylated and unmethylated cytosine. The unmethylated cytosine is converted to uracil, while the methylated one is not changed. During PCR, the methylated CpGs are converted to guanine and the unmethylated ones are converted to adenine. The SAP reaction step is needed to prevent embedding of remaining dNTP during the iPlex reaction. On the iPlex reaction step, the extension primer elongates by one terminating nucleotide, and then the resulting fragments having different masses are analyzed on a mass spectrometer.

The methylation level was determined by calculating the ratio of peaks corresponding to primer extension products. In the case of CpGs being methylated, the primer was extended with cytosine, while, in the unmethylated state, it was extended with thymine. The methylation level was calculated using the formula: A/(A + B) × 100%, where A represents the relative intensity of the cytosine peak, and B represents the relative intensity of the thymine peak.

In our study, the DNA methylation assessment protocol was optimized after an initial assessment of the repeatability of the obtained results. The final protocol was committed only after achieving intraclass correlation coefficient (ICC) greater than 0.9 (calculated in R using the icc() function).