Comparison of Methods for Quantification of Global DNA Methylation

DNA methylation, particularly the methylation of cytosine residues at the C-5 position (5-methylcytosine, or 5-mC), plays a pivotal role in the regulation of gene expression and various cellular processes. Aberrancies in the epigenome have been implicated in human disease, including cancer. Thus, quantification of global DNA methylation levels is essential for understanding epigenetic alterations associated with physiological and pathological conditions. Several methods have garnered commonplace usage in research labs for quantifying global DNA methylation, each with its own unique working principle, advantages, and disadvantages.

HPLC/MS

Considered “the gold standard” for quantitative analysis of modified nucleic acid bases, high performance liquid chromatography together with mass spectrometry (HPLC/MS) has been routinely applied in the quantification of DNA methylation due to its high accuracy, precision, and sensitivity. A typical HPCL/MS protocol initially involves nuclease P1 and alkaline phosphatase digestion of isolated DNA. Following digested DNA purification, deoxyribonucleosides are separated by reverse phase HPLC on a C18 column. Deoxyribonucleoside mass chromatograms are then acquired by tandem mass spectrometry, and the target modification is quantified based on a standard curve of pure modified deoxyribonucleoside standards. Although highly quantitative and reproducible, HPLC/MS requires expensive instrumentation, long and time-consuming protocols, specialized technical expertise, and trained personnel.

Bisulfite Conversion

Chemical treatment of DNA that differentially targets methylated and unmethylated cytosine residues is another popular option, and can be accompanied downstream by sequencing for base resolution analysis of genome-wide DNA methylation (Whole Genome Bisulfite Sequencing). Treating DNA with bisulfite, followed by PCR amplification, cloning, and sequencing, yields reliable information about DNA cytosine methylation states. Bisulfite conversion is a chemical reaction whereby unmethylated cytosines are deaminated to uracil while 5-mC is left intact. Consequently, methylated and unmethylated DNA cytosine residues are differentiated from one another. However, the traditional conversion method requires a lengthy protocol time that leads to heavy DNA degradation, high inappropriate 5-mC deamination, and low cytosine conversion rate.

DNA conversion via bisulfite treatment.

ELISA

DNA methylation enzyme-linked immunosorbent assays (ELISAs) provide a simple, convenient, and affordable way to rapidly assess global levels of a particular methylated DNA target, before pursuing the more in-depth and costly aforementioned approaches. Such ELISA-based methods use antibodies to detect and quantify a specific DNA methylation modification within DNA samples. These assays typically involve immobilizing DNA on a solid surface, like a microplate, and then applying the antibody for detection. While analogous in terms of assay workflow (e.g., sample binding to assay wells, target capture by primary antibody, signal detection with enzyme-conjugated secondary antibody), compared with conventional ELISAs, DNA methylation ELISAs are extremely sensitive with much lower detection limits, allowing for the assessment of a wider range of sample types.

Working principle of EpigenTek’s MethylFlash™ ELISA-based assay for global 5-mC DNA methylation quantification.

Maximizing your budget

Whether the target is 5-mC or 5-hydroxymethylcytosine (5-hmC), DNA methylation ELISAs offer a high-throughput, cost-effective means of analyzing a variety of modified DNA forms. As pioneers in the R&D of epigenetics-based research tools, EpigenTek has leveraged its proprietary MethylFlash technology to develop rapid, accurate, and highly cited quantitation immunoassays for global DNA methylation analysis. The MethylFlash™ Global DNA Methylation (5-mC) ELISA Easy Kit (catalog #P-1030) and the MethylFlash™ Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit (catalog #P-1032) have been specially designed to use fully intact input DNA as starting material, eliminating the need for additional sample processing (e.g., denaturation, fragmentation) required by similar assays. These kits combine the convenience and speed of ELISA with high sensitivity, specificity, universality, accuracy, and flexibility, making them valuable tools for researchers studying global DNA methylation in various contexts.