SpeedRun One-Hour 5-hmC ELISA Kit (Colorimetric)

The SpeedRun™ One-Hour 5-hmC ELISA Kit (Colorimetric) is a complete set of optimized reagents designed for detecting global DNA hydroxymethylation levels using DNA isolated from any species including mammals, plants, fungi, bacteria, and viruses in a variety of forms including, but not limited to, cultured cells, fresh and frozen tissues, paraffin-embedded tissues, plasma/serum samples, and body fluid samples. Both single stranded DNA and double stranded DNA with a size of 200 bps to full length are suitable for use. The kit has the following advantages and features:

• Fast: Reduced steps so that the entire procedure only needs 1 hour*
• Robust: Improved kit composition allows the assay to have a greater “signal window” with reduced variation between replicates
• Convenient: Inherently low background noise, thereby eliminating the need for DNA denaturation and plate blocking steps
• Sensitive: Detection limit can be as low as 0.01% hydroxymethylated DNA from 100 ng of input DNA
• Specific: High specificity to 5-hmC, with no cross-reactivity to unmethylated cytosine or methylated cytosine within the indicated concentration range of the sample DNA
• Universal: Positive and negative controls allow detection of DNA hydroxymethylation in any species from either single-stranded or double-stranded input DNA
• Flexible: Strip-well microplate format makes the assay available for manual or high-throughput analysis

Background Information
5-hydroxymethylcytosine (5-hmC), as a sixth DNA base with functions in transcription regulation, has been detected to be abundant in human and mouse brains and embryonic stem (ES) cells. In mammals, it can be generated by the oxidation of 5-mC, a reaction mediated by the ten-eleven translocation (TET) family of 5-mC-hydroxylases. 5-hmC has been demonstrated to be tissue specific, ranging from undetectable levels in cultured cell lines to 0.6% in human brain tissues, and can be as high as 8% of total DNA in some other species. The biological significance of 5-hmC as an important epigenetic modification in phenotype and gene expression has been recently recognized. For example, global decrease in 5-hmC content (DNA hypo-hydroxymethylation) has been exhibited in nearly all cancers and has been proposed as a molecular marker and therapeutic target in cancer as well. Accurate and convenient detection of 5-hmC would be extremely useful for identifying and understanding the global hydroxymethylation changes that occur in DNA during various physiological and pathological processes such as in cancer. To address this, EpigenTek offers a series of Hydroxymethylated DNA Quantification Kits to quantify 5-hmC or hydroxymethylated DNA and further refines its hydroxymethylated DNA assay expertise with the development of the SpeedRun™ One-Hour 5-hmC ELISA Kit (Colorimetric).

Principle & Procedure
This kit contains all reagents necessary for the quantification of global DNA hydroxymethylation. In this assay, DNA is bound to strip-wells with a high binding buffer. The hydroxymethylated fraction of DNA is detected using capture and detection antibodies and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer. The percentage of hydroxymethylated DNA is proportional to the OD intensity measured.

Starting Materials
Input materials can be either single-stranded or double-stranded DNA, with a size of 200 bps to full length. The amount of DNA for each assay can range from 50 to 200 ng. For optimal quantification, 100 ng of input DNA is recommended, as hydroxymethylated DNA varies from tissue to tissue and can be less than 0.6% of total DNA in some species.