The MethylFlash™ Global DNA Methylation (5-mC) ELISA Easy Kit  is a complete set of optimized buffers and reagents to colorimetrically quantify global DNA methylation status by specifically measuring total levels of 5-methylcytosine (5-mC) in a simplified, "one-step" ELISA-like reaction. This allows global DNA methylation to be directly measured more quickly than other techniques such as LUMA, LINE-1, Alu or LTR-based assays. As a fourth generation technology of EpigenTek's patented global DNA methylation technique, it is a further refinement of the popular, predecessor MethylFlash kit by improving upon speed, simplicity, sensitivity, and reproducibility.
This kit is also specifically optimized for paired use with the MethylFlash Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit (Colorimetric) for simultaneously quantifying both methylated DNA and hydroxymethylated DNA.
This kit has the following advantages and features:
- Fast - Reduced steps so that the entire procedure only needs 2 hours
- Robust - Improved kit composition allows the assay to have a greater "signal window" with reduced variation between replicates
- Convenient - Inherently low background noise, thereby eliminating the need for DNA denaturation and plate blocking steps
- Sensitive - Detection limit can be as low as 0.05% methylated DNA from 100 ng of input DNA
- Specific -Â High specificity to 5-mC, with no cross-reactivity to unmethylated cytosine and no cross-reactivity to hydroxymethylcytosine within the indicated concentration range of the sample DNA
- Universal - Positive and negative controls are included and allow for detection of DNA methylation in any species from either single-stranded or double-stranded input DNA
- Accurate -Â Optimized positive controls that can be fractionalized in percentage scale, allowing the assay to be more accurate and highly comparable with HPLC-MS analysis
- Flexible - Strip-well microplate format makes the assay available for manual or high throughput analysis
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Background
DNA methylation occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring by DNA methyltransferases, resulting in 5-methylcytosine (5-mC). In somatic cells, 5-mC is found almost exclusively in the context of paired symmetrical methylation of the dinucleotide CpG, whereas in embryonic stem (ES) cells, a substantial amount of 5-mC is also observed in non-CpG contexts. Levels of 5-mC are variable in animal genomes, ranging from undetectable amounts in some insects to about 2% of total DNA in vertebrates. The level of 5-mC in plants generally accounts for 0.5-2% and can be as high as 8% of total DNA in some other species. The biological importance of 5-mC as a major epigenetic modification in phenotype and gene expression has been recognized widely. For example, global decrease in 5-mC content (DNA hypomethylation) is likely caused by methyl-deficiency due to a variety of environmental influences, and has been proposed as a molecular marker in multiple biological processes such as cancer. It has been well demonstrated that the decrease in global DNA methylation is one of the most important characteristics of cancer. A few novel modified nucleotides, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) have been detected in human and mouse tissues as well as embryonic stem cells. In mammals, these modified nucleotides can be generated by iterative oxidation of 5-methylcytosine, a reaction mediated by the TET family of enzymes.
Principle & Procedure
This kit contains all reagents necessary for the quantification of global DNA methylation. In this assay, DNA is bound to strip-wells that are specifically treated to have a high DNA affinity. The methylated fraction of DNA is detected using capture and detection antibodies and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer. The percentage of methylated DNA is proportional to the OD intensity measured.
Starting Materials
Input DNA should be relatively pure with 260/280 ratio >1.6 and can be diluted with water or TE buffer. The DNA amount can range from 5 ng to 200 ng per reaction. However, we recommend using 100 ng of DNA, which is the optimized input amount for the best results. DNA can be isolated from any species such as mammals, plants, fungi, bacteria, and viruses in a variety of forms including, but not limited to, cultured cells, fresh and frozen tissues, paraffin-embedded tissues, plasma/serum samples, and body fluid samples. Both single stranded DNA and double stranded DNA with a size of 200 bps to full length is suitable for use.
EpigenTek vs. Competitors: Performance Comparison
| Feature |
EpigenTek |
Competitor Z |
Competitor C |
| Protocol Time |
2 hours |
>3 hours |
>3 hours |
| Sample Input Type |
Isolated DNA is directly used for the assay; no blocking needed
|
Isolated DNA requires single strand conversion; blocking needed
|
Urine; Isolated DNA (requires single strand conversion, nuclease digestion, and alkaline phosphatase treatment)
|
| Input Amount |
100 ng optimal |
100 ng optimal |
2 µg of DNA minimum per assay |
| Sensitivity |
LOD 0.05% |
LOD >0.5% |
150 nM of 5-mC deoxynucleoside |
| Controls |
Synthesized PC (methylated) and NC (unmethylated) oligos
|
PC and NC E. coli gDNA (require single strand conversion); PC methylated with CpG Methylase
|
5-mC deoxynucleoside standard
|
| Suitability for Various Samples |
Universal for any species |
Only suitable for a few species |
Limited to specific species |
| Methylation Quantification |
Both relative and absolute |
Only relative |
Only relative |
| Accurately Reflects True Methylation Status |
High |
Low |
Low |
| Citations |
Very high
>780
|
Low
~120
|
Low
|
Our single B-cell derived recombinant rabbit monoclonal antibodies have vastly improved reliability and reproducibility.
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![5-Methylcytosine (5-mC) Monoclonal Antibody [33D3]](images/a1014data1.gif)
