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We recommend using a newer version of this product: MethylFlash Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit (Colorimetric) 

The MethylFlash™ Hydroxymethylated DNA Quantification Kit (Colorimetric) is a complete set of optimized buffers and reagents to colorimetrically quantify global DNA hydroxymethylation by specifically measuring levels of 5-hydroxymethylcytosine (5-hmC) without cross-reactivity to methylcytosine and unmethylated cytosine, in an ELISA-like microplate-based format.

This kit is also specifically optimized for paired use with the MethylFlash Methylated DNA Quantification Kit (Colorimetric) for simultaneously quantifying both methylated DNA and hydroxymethylated DNA.

About 5-Hydroxymethylcytosine
5-hmC is a modified form of cytosine, recently discovered in animal tissues. The function of 5-hmC in epigenetics may be different from its forerunner 5-methylcytosine (5-mC) and currently remains a mystery. It is believed, though, that 5-hmC plays an important role in switching genes on and off.  The presence of 5-hmC makes it necessary to not only re-evaluate existing DNA methylation data, but also necessary to determine the relative distribution and changes of 5-hmC in human tissues of healthy and diseased statuses. Prior to Epigentek’s MethylFlash™ technology, there were no methods that could be used for practically and routinely identifying 5-hmC and discriminating this base from 5-mC.

Distinguishing Between 5-hmC and 5-mC
Currently used methylated DNA analysis methods including restriction enzyme digestion and bisulfite or MeDIP-mediated MS-PCR and sequencing are not suitable for 5-hmC detection as 5-hmC and 5-mC are virtually indistinguishable between each other with these methods. To address this problem, Epigentek offers the MethylFlash™ Hydroxymethylated DNA Quantification Kit which uses a unique procedure to quantify global DNA hydroxymethylation. This product provides a cost-effective way to measure levels of 5-hydroxymethylcytosine and to distinguish between 5hmC, 5mC, and C. This allows for researchers to re-evaluate their DNA methylation data for DNA hydroxymethylation and to efficiently look for DNA hydroxymethylation in new DNA samples.  The kit has the following advantages and features:

• Colorimetric assay with easy-to-follow steps for convenience and speed. The entire procedure can be completed within 3 hours and 45 minutes.
• High sensitivity, of which the detection limit can be as low as 40 pg of hydroxymethylated DNA and accepts as low as 50 ng of input genomic DNA.
• High specificity with no cross-reactivity to methylcytosine and unmethylated cytosine. 5-hydroxymethylcytosine is separately detected. 
• Universal positive and negative controls are included, which are suitable for quantifying hydroxymethylated DNA from any species.
• Strip-well microplate format makes the assay flexible for either manual or high throughput analysis.
• Simple, reliable, and consistent assay conditions.

Comparison of Available ELISA-based Global DNA Methylation Assay Methods