The MethylFlash ™ Methylated DNA Quantitation Kit (Colorimetric) is a comprehensive set of buffers and reagents optimized to quantify overall DNA methylation colorimetrically by specifically measuring 5-methylcytosine (5-mC) levels in an ELISA-like microplate format. Compared to LUMA or LINE-1, Alu, and LTR-based assays, MethylFlash ™ technology directly quantifies true global DNA methylation. This kit is also specifically optimized for use in conjunction with the MethylFlash Hydroxymethylated DNA Quantitation Kit (colorimetric) to simultaneously quantify both methylated and hydroxymethylated DNA.
The MethylFlash ™ Methylated DNA Quantitation Kit (Colorimetric) is a further enhancement to our previous Methylamp ™ Global DNA Methylation Quantitation Kits by simplifying workflow and improving the consistency of results. It uses an innovative method to allow background signals to be extremely low, eliminating plate drying and blocking steps. Compared to chromatography-based methods such as HPLC or mass spectrometry, this product provides a cost-effective way to accurately measure 5-methylcytosine levels.
The kit has the following advantages and features:
- Colorimetric assay with easy-to-follow steps for convenience and speed. The whole procedure can be finished in 4 hours.
- The innovative composition of the kit allows for extremely low background signals, eliminating the need to block the plate and allowing the assay to be simple, accurate, reliable, and consistent.
- High sensitivity, whose detection limit can be as low as 0.2 ng of methylated DNA and accepts as little as 50 ng of input genomic DNA.
- Optimized antibody and enhancer solutions allow for high specificity at 5mC, no cross-reactivity with unmethylated cytosine, and no cross-reactivity or low reactivity with hydroxymethylcytosine within the indicated concentration range of the sample DNA.
- Universal positive and negative controls are included, which are suitable for quantifying methylated DNA of any species.
- The strip-well microplate format makes the assay flexible: manual or high-throughput testing.
Principle and procedure
The MethylFlash ™ Methylated DNA Quantitation Kit (Colorimetric) contains all the reagents necessary for the quantification of overall DNA methylation. In this assay, DNA is bound to wells that are specifically treated to have a high affinity for DNA. The methylated fraction of DNA is detected using capture and detection antibodies and then quantitated by an ELISA-like reaction by reading absorbance on a microplate spectrophotometer at 450 nm. The amount of methylated DNA is proportional to the measured OD intensity, which can be calculated with the formulas included in the kit for the relative methylation status of two different DNA samples or the absolute quantification of 5-methylcytosine (5mC) using a standard curve.
Easy, fast, and flexible
The entire colorimetric assay has easy-to-follow steps for convenience and speed, which can be completed in 4 hours. The strip well microplate format allows flexible assay in manual or high throughput analysis. The kit includes positive and negative universal controls to quantify methylated DNA of any species, such as mammals, plants, fungi, bacteria, and viruses.
Safe and convenient
All necessary reagents, including negative controls and positive controls, for the quantification of overall DNA methylation, are conveniently packaged in the kit. Direct colorimetric quantification of DNA samples replaces outdated 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 enable accurate quantification of methylated DNA with high sensitivity and specificity. The limit of detection of input DNA can be as low as 0.2 ng of methylated DNA.