MethylFlash Hydroxymethylated DNA (5-hmC) Quantification Kit (Fluorometric)

Product Description

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 5-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 5-hmC, 5-mC, 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.

Advantages

• 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.
• 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

Principle & Procedure

In this assay, DNA is bound to strip wells that are specifically treated to have a high DNA affinity. The hydroxymethylated fraction of DNA is detected using capture and detection antibodies and then quantified fluorometrically by reading the RFU (relative fluorescence units) in a fluorescence microplate spectrophotometer at excitation 530 +/-20 nm and emission 580 +/-20 nm. The amount of hydroxymethylated DNA is proportional to the fluorescence intensity measured, which can be calculated with the kit's included formulas for relative hydroxymethylation status of two different DNA samples or absolute quantification of 5-hydroxymethylcytosine (5-hmC) using a standard curve.

Easy, Fast, and Flexible

The entire fluorometric assay has easy-to-follow steps for convenience and speed, which can be completed in 3 hours and 20 minutes. The strip-well microplate format allows for a flexible assay in manual or high throughput analysis. Universal positive and negative controls are included with the kit for quantifying hydroxymethylated DNA from any species such as mammals, plants, fungi, bacteria, and viruses.

Safe and Convenient

All the needed reagents, including negative controls and positive controls, for quantification of global DNA hydroxymethylation are conveniently packaged in the kit. The direct fluorometric quantification of DNA samples replaces obsolete or inferior methods and eliminates the need for DNA digestion/denaturation, radioactivity, extraction, or chromatography.'

Highly Sensitive and Specific

The novel procedure and proprietary kit compositions allow for accurate quantification of hydroxymethylated DNA to be achieved with high sensitivity and specificity. The detection limit of the input DNA can be as low as 10 pg of hydroxymethylated DNA with a dynamic range from 10 pg to 5 ng (see Fig. 2). Analytic sensitivity is about ten-fold higher than that obtained by HPLC (0.008% vs 0.08%). There is no cross-reactivity to methylcytosine and unmethylated cytosine so that only hydroxymethylated DNA (5-hmC) is detected.

Demonstration of high sensitivity and specificity of 5-hydroxymethylcytosine detection achieved by the MethylFlash kit. Synthetic unmethylated DNA (contains only 5-cytosine), methylated DNA (contains only 5-methylcytosine), and hydroxymethylated DNA standard (contains only 5-hydroxymethylcytosine) were added into the assay wells at different concentrations and then measured with the MethylFlash Hydroxymethylated DNA Quantification Kit (Fluorometric).

Responsive, Reliable, and Practical

Based on its working principle and the microplate format, the kit can be practically and routinely used for any species from a variety of forms including from cultured cells, fresh and frozen tissues, paraffin-embedded tissues, plasma/serum samples, and body fluid samples.