The MethylFlash ™ Methylated DNA Quantitation Kit (Fluorometric) is a complete set of buffers and reagents optimized to quantify overall DNA methylation fluorometrically by specifically measuring 5-methylcytosine (5-mC) levels in a 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 (Fluorometric) to simultaneously quantify both methylated and hydroxymethylated DNA.
The MethylFlash ™ Methylated DNA Quantitation Kit (Fluorometric) is a further enhancement to our previous SuperSense ™ 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:
- Fluorometric 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 50 pg of methylated DNA and accepts as little as 20 ng of input genomic DNA
- Optimized antibody and enhancer solutions allow high specificity at 5-mC, no cross-reactivity with unmethylated cytosine, and no or negligible cross-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 (Fluorometric) 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 is then quantified fluorometrically by reading the absorbance on a fluorescence microplate spectrophotometer at 530 excitations and 590 nm emission.
The amount of methylated DNA is proportional to the measured RFUs (Relative Fluorescence Units), 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 ( 5-mC) using a standard curve.
Easy, fast, and flexible
The entire fluorometric 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 fluorometric 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 allow accurate quantification of methylated DNA with high sensitivity and specificity. The limit of detection of input DNA can be as low as 50 pg of methylated DNA.