The MethylFlash Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit (Colorimetric) is a complete set of buffers and reagents optimized to colorimetrically quantify the hydroxymethylation status of global DNA by specifically measuring 5-hydroxymethylcytosine (5-hmC) levels. ) in a simplified way, Format similar to the “one-step” ELISA.
As the fourth generation technology of Epigentek’s popular global DNA methylation technique, it is a further refinement of the predecessor MethylFlash kit by improving speed, simplicity, sensitivity, and reproducibility. This kit is also specifically optimized for use in conjunction with the MethylFlash Global DNA Methylation (5-mC) ELISA Easy (colorimetric) kit to simultaneously quantify both methylated and hydroxymethylated DNA.
This kit has the following advantages and features:
- Fast – reduced steps so the entire procedure only takes 2 hours
- Robust: the improved composition of the kit allows the assay to have a larger “signal window” with reduced variation between replicates
- Convenient – Inherently low background noise, eliminating the need for DNA denaturation and plaque blocking steps
- Sensitive: the detection limit can be as low as 0.01% hydroxymethylated DNA from 100 ng of input DNA
- Specific: high specificity for 5-hmC, no cross-reactivity with unmethylated cytosine or methylated cytosine within the indicated concentration range of the sample DNA
- Universal: positive and negative controls that allow the detection of DNA hydroxymethylation in any species from single-stranded or double-stranded input DNA
- Accurate – Optimized positive controls that can be fractionated on a percentage scale, allowing the assay to be more accurate and highly comparable with HPLC-MS analysis
- Flexible – Strip-well microplate format makes the assay available for manual or high-throughput analysis based on a single sample duplicate assay
5-Hydroxymethylcytosine (5-hmC), as a sixth base of DNA with functions in the regulation of transcription, has been found to be abundant in human and mouse brains and in embryonic stem cells (ES). In mammals, it can be generated by oxidation of 5-mC, a reaction mediated by the ten-eleven translocation (TET) family of 5-mC-hydroxylases. 5-hmC has been shown to be tissue-specific, ranging from undetectable levels in cultured cell lines to 0.6% in human brain tissues, and can reach 8% of total DNA in some other species.
The biological importance of 5-hmC has recently been recognized as an important epigenetic modification in phenotype and gene expression. For example, the global decrease in 5-hmC content (DNA hypomethylation) has been shown in almost all cancers and has also been proposed as a molecular marker and therapeutic target in cancer.
Principle and procedure
This kit contains all the reagents necessary for the quantification of the overall hydroxymethylation of DNA. In this assay, DNA is bound to strip wells that are specifically treated to have a high affinity for DNA. The hydroxymethylated fraction of DNA is detected using a 5-hmC mAb-based detection complex in a one-step fashion and then quantified colorimetrically by reading absorbance on a microplate spectrophotometer. The percentage of hydroxymethylated DNA is proportional to the measured DO intensity.
Materials to get started
The input DNA should be relatively pure with a ratio 260/280> 1.6 and can be diluted with water or TE buffer. The amount of DNA can range from 20 ng to 200 ng per reaction. However, we recommend using 100 ng of DNA, which is the optimized input amount for the best results.