Principle & Procedure
The MethylFlash™  Hydroxymethylated DNA Quantification Kit (Fluorometric) contains all reagents necessary for the fluorometric quantification of global DNA hydroxymethylation. 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 nm and emission 590 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.

Fig. 2. Demonstration of high sensitivity and specificity of 5-hydroxymethylcytosine detection achieved by the MethylFlash™ kit. Synthetic unmethylated DNA (contains only 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).

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.

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. To demonstrate the capabilities of the kit, it has been successfully used for quantifying the content of 5-hmC in DNA from mouse brains and HeLa cells. The percentage of 5-hmC measured by the MethylFlash™ kit is parallel and comparable to that detected by HPLC and LC-MS methods (see Table 1). Through the use of the kit, a high abundance of 5-hmC in normal human brains and colon tissues is discovered for the first time. Furthermore, it shows that 5-hmC accounts for 32% and 16% of total methylated cytosine (5-mC plus 5-hmC) in normal human brains and colon tissues, respectively (see Table 2). In contrast, 5-hmC is low or absent in colon cancer cell lines (HCT116) and cervical cancer cell lines (HeLa).

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