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

The SpeedRun™ One-Hour 5-mC ELISA Kit (Colorimetric) is a complete set of optimized reagents designed for detecting and quantifying global DNA methylation levels using DNA isolated from any species, including mammals, plants, fungi, bacteria, and viruses. The kit can be used with DNA from a variety of sample types, including cultured cells, fresh and frozen tissues, paraffin-embedded tissues, plasma/serum samples, and body fluid samples. Both single-stranded 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 allow the entire procedure to be completed in just 1 hour*
• Robust: Improved kit composition provides a greater signal window with reduced variation between replicates
• Convenient: Inherently low background noise eliminates the need for DNA denaturation and plate blocking steps
• Sensitive: Detection limit can be as low as 0.05% methylated DNA from 100 ng of input DNA
• Specific: High specificity to 5-mC, with no cross-reactivity to unmethylated cytosine or hydroxymethylated cytosine within the indicated concentration range of the sample DNA
• Universal: Positive and negative controls are included and allow detection of DNA methylation in any species using either single-stranded or double-stranded DNA
• Flexible: Strip-well microplate format allows manual or high-throughput analysis

Background Information
DNA methylation occurs through the covalent addition of a methyl group at the 5-carbon of the cytosine ring by DNA methyltransferases, resulting in 5-methylcytosine (5-mC). Levels of 5-mC vary among species, ranging from undetectable amounts in some insects to approximately 2% of total DNA in vertebrates. In plants, 5-mC generally accounts for 0.5-2% of total DNA and can be as high as 8% in some other species. The biological importance of 5-mC as a major epigenetic modification involved in phenotype and gene expression has been widely recognized. A global decrease in 5-mC content, or DNA hypomethylation, has been associated with a variety of environmental influences and biological processes, including cancer. Accurate and convenient detection of 5-mC can help identify and characterize global DNA methylation changes that occur during physiological and pathological processes. To address this need, EpigenTek offers a series of Methylated DNA Quantification Kits to quantify 5-mC or methylated DNA and further refines its methylated DNA assay expertise with the development of the SpeedRun™ One-Hour 5-mC ELISA Kit (Colorimetric).

Principle & Procedure
The kit contains all reagents necessary for the quantification of global DNA methylation. In this assay, DNA is bound to strip-wells with a high binding buffer. The methylated fraction of DNA is detected using 5-mC Detection Complex Solution and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer. The percentage of methylated DNA is proportional to the OD intensity measured.

Starting Materials
Input materials can be single-stranded or double-stranded DNA isolated from any species, with a size of 200 bps to full length. The amount of DNA for each assay can range from 20 to 200 ng, with 100 ng recommended for optimal quantification.