The MethylFlash ™ Global DNA Methylation (5-mC) ELISA Easy Kit is a complete set of buffers and reagents optimized to colorimetrically quantify the global methylation status of DNA by specifically measuring 5-methylcytosine (5-mC) levels in a simplified format. , “one-step” reaction similar to ELISA. This makes it possible to directly measure global DNA methylation faster than other techniques such as LUMA, LINE-1, Alu, or LTR-based assays.
As a fourth-generation technology in EpigenTek’s proprietary global DNA methylation technique, it is a further refinement of the popular MethylFlash predecessor kit by improving speed, simplicity, sensitivity, and reproducibility. This kit is also specifically optimized for use in conjunction with the MethylFlash Global DNA Hydroxymethylation (5-hmC) ELISA Easy (colorimetric) kit to simultaneously quantify both methylated and hydroxymethylated DNA.
This kit has the following advantages and features:
- Fast: reduced steps so that the entire procedure only takes 2 hours
- Robust: the improved composition of the kit allows the assay to have a larger “signal window” with reduced variation between replicates
- Convenient – Inherently low background noise, eliminating the need for DNA denaturation and plaque blocking steps
- Sensitive: the limit of detection can be as low as 0.05% of methylated DNA from 100 ng of input DNA
- Specific: high specificity for 5-mC, no cross-reactivity with unmethylated cytosine, and no cross-reactivity with hydroxymethylcytosine within the indicated concentration range of the sample DNA
- Universal: Positive and negative controls are included that allow detection of DNA methylation in any species from single-stranded or double-stranded input DNA
- Accurate – Optimized positive controls that can be fractionated on a 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
DNA methylation occurs by the covalent addition of a methyl group at carbon 5 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 symmetric methylation of CpG dinucleotide, whereas in embryonic stem cells (ES), a substantial amount of 5-mC is also observed in non-CpG contexts. . Levels of 5-mC are variable in animal genomes, from undetectable amounts in some insects to about 2% of total DNA invertebrates.
The 5-mC level in plants generally represents 0.5-2% and can be as high as 8% of the total DNA in some other species. The biological importance of 5-mC as an important epigenetic modification in phenotype and gene expression has been widely recognized. For example, the global decrease in 5-mC content (DNA hypomethylation) is likely caused by methyl deficiency due to a variety of environmental influences, and it has been proposed as a molecular marker in multiple biological processes such as cancer.
It is well established that the decrease in overall DNA methylation is one of the most important characteristics of cancer. Some new modified nucleotides, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxytosine (5-caC) have been detected in human and mouse tissues, as well as in 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 and procedure
This kit contains all the 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 affinity for DNA. The methylated fraction of DNA is detected using capture and detection antibodies and is then quantified colorimetrically by reading the absorbance on a microplate spectrophotometer. The percentage of methylated DNA is proportional to the intensity of OD measured.
Materials to get started
The input DNA must be relatively pure with a ratio 260/280> 1.6 and can be diluted with water or TE buffer. The amount of DNA can range from 20 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 samples. of bodily fluids. Both single-stranded DNA and double-stranded DNA with a size of 200 bps at full length are suitable for use.