Product Details
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 +/-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.
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 conrols 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-reactivit 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 brais 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).
| | MethylFlash | HPLC1 | LC-MS2 | | Mouse Brain | 0.16% | 0.55% | 0.13% | | HeLa Cells | <0.02% | undetectable | not determined |
Table 1. Comparison of the MethylFlash™ Hydroxymethylated DNA Quantification Kit and chromatography-based methods for the detection of 5-hydroxymethylcytosine show in percentage of 5-hmC present in two DNA sample types. 1 Kriaucionus S et al: Science 324: 929-930 2009
2 Munzel M et al: Angewandte Chemie 122: ePub 2010 | | | % 5-hmC | % 5-mC | 5-hmC/(5-mC + 5-hmC) | | Human Brain | 0.67% | 1.00% | 40.00% | | Mouse Brain | 0.16% | 1.53% | 10.00% | | Human Placenta | 0.06% | 0.67% | 8.00% | | Human Colon | 0.47% | 1.46% | 24.00% | | HCT116 | <0.01% | 1.30% | <1.00% | | HeLa | <0.02% | 1.62% | <1.00% |
 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.
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Product Components
HF1 (10X Wash Buffer)
HF2 (Binding Solution)
HF3 (Negative Control I 20 µg/ml)*
HF4 (Negative Control II 20 µg/ml)*
HF5 (Positive Control 20 µg/ml)*
HF6 (Capture Antibody 1000 µg/ml*
HF7 (Detection Antibody 400 µg/ml)*
HF8 (Enhancer Solution)*
HF9 (Fluoro Developer)*
HF10 (Fluoro Enhancer)*
HF11 (Fluoro Dilutor)
8-Well Assay Strips (With Frame)
User Guide
* Spin the solution down to the bottom prior to use.
Note: The HF3 Negative Control I is an nmethylated polynucleotide containing 20% of cytosine. The HF4 Negative Control II is a methylated polynucleotide containing 20% of 5-methylcytosine. The HF5 Positive Control is a hydroxymethylated polynucleotide containing 20% of hydroxymethylcytosine.
