| Assay/References |
DNA preparation/treatment |
Strengths |
Limitations |
Other comments |
| High performance liquid chromatography (HPLC) [29] |
Total genomic DNA hydrolysed to deoxyribonucleotides and treated with ribonuclease to remove contaminating RNA. Deoxyribonucleotides converted to deoxyribonucleosides (treat with alkaline phosphatase). Products separated by reverse phase HPLC. Quantification with external standards or UV absorbance at 254nm. |
Highly quantitative and reproducible |
Requires large amounts of DNA (several micrograms).
Not suitable for high throughput.
Contaminants may co-elute with DNA and impact on accuracy. |
Has been the preferred quantitative technique for determining global DNA methylation prior to the development of LC-MS/MS assays |
| M.SssI acceptance assay [16,30] |
Genomic DNA is incubated with M.SssI DNA methyltransferase in the presence of tritiated S-adenosylmethionine and immobilised on nitrocellulose paper. Radiation is measured in scintillation counter. |
Simple and rapid procedure. |
High inter-assay variability. Semi-quantitative |
Difficult to compare results with those generated by other laboratories. Requires radiation. |
| Enzyme immunoassay Epigenetek (P-1034); Cell Biolabs (STA-380 and STA-381) |
Genomic DNA is denatured, immobilized and incubated with monoclonal antibodies against 5mC. Levels of 5mC determined by intensity of staining determined by incubation with fluorescein-conjugated secondary antibody. |
Can be used to map the location of DNA methylation on chromosomes in individual cells e.g. juxtacentromeric regions. |
Qualitative assay. High inter-assay variability. |
Commercially available kits |
| Luminometric methylation assay (LUMA) [14] |
Based on combined DNA cleavage by methylation-sensitive restriction enzymes and polymerase extension assay by pyrosequencing |
Sensitive (requires 200-500 ng DNA), semi-quantitative |
Assay is sequence-specific and may not truly represent global DNA methylation levels |
Difficult to compare results with those generated by other methods |
| Bisulfite sequencing of repetitive elements [21,31,32] |
Bisulfite treatment of DNA and real-time PCR or standard PCR amplification of repetitive DNA elements (using combined bisulfite restriction analysis [COBRA] or pyrosequencing) |
Very sensitive (<100 ng), high-throughput, relatively quantitative for each repeat element |
May not truly represent global DNA methylation levels. High inter-assay variability, bisulfite conversion of DNA introduces errors due to spontaneous deamination of methylated cytosines into thymine which occur frequently at repetitive elements |
Very popular assay for applications where only small quantities of DNA are available. |
| End-specific PCR [33] |
Quantitative assessment of the level of unmethylated LINE-1, Alu and LTR elements using methylation-sensitive restriction enzyme digestion and real-time PCR with a fluorescence-based readout |
Ultra sensitive (only 1-5ng needed), high-throughput. |
Surrogate for global DNA methylation. Only able to analyze specific subsets of LINE and Alu repeats. Assay is only suitable for human DNA samples. Semi-quantitative. |
Useful when only extremely small quantities of DNA are available. |
| LC-MS/MS [34] |
Identical to HPLC except quantitation is performed using spiked internal standards and measured using mass spectrometry |
Sensitive (100-500 ng), quantitative, reproducible, absolute quantitation |
High instrument setup costs, expensive to synthesize or purchase internal standards, requires mass spectrometry expertise |
Considered the gold standard method. |
