MethylFlash Hydroxymethylated DNA Quantification Kit (Fluorometric)

The MethylFlash™ Hydroxymethylated DNA Quantification Kit (Fluorometric) is a complete set of optimized buffers and reagents to fluorometrically quantify global DNA hydroxymethylation by specifically measuring levels of 5-hydroxymethylcytosine (5-hmC) without cross-reactivity to methylcytosine and unmethylated cytosine.

This kit is also specifically optimized for paired use with the MethylFlash Methylated DNA Quantification Kit (Fluorometric) 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 (Fluorometric) 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:

• Fluorometric assay with easy-to-follow steps for convenience and speed. The entire procedure can be completed within 3 hours and 20 minutes.
• High sensitivity, of which the detection limit can be as low as 10 pg of hydroxymethylated DNA and accepts as low as 20 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 

Table 1. Comparison of the MethylFlash™ Hydroxymethylated DNA Quantification Kit and chromatography-based methods for the detection of 5-hydroxymethylcytosine, shown in percentage of 5-hmC present in two DNA sample types.

¹ Munzel M et al: Angewandte Chemie, 122: ePub, 2010

Table 2. Percentage of 5-hmC and 5-mC in different tissues/cells, measured with the MethylFlash™ Hydroxymethylated DNA Quantification Kit and the MethylFlash™ Methylated DNA Quantification Kit, respectively.