The MethylFlash™ Methylated DNA Quantification Kit (Fluorometric) is a complete set of optimized buffers and reagents to fluorometrically quantify global DNA methylation by specifically measuring levels of 5-methylcytosine (5-mC) in a microplate-based format. Compared to LUMA or LINE-1, Alu, and LTR-based assays, MethylFlash™ technology directly quantifies actual global DNA methylation.
This kit is also specifically optimized for paired use with the MethylFlash Hydroxymethylated DNA Quantification Kit (Fluorometric) for simultaneously quantifying both methylated DNA and hydroxymethylated DNA.
The MethylFlash™ Methylated DNA Quantification Kit (Fluorometric) is a further refinement of our previous SuperSense™ global DNA methylation quantification kits by simplifying the workflow and improving the consistency of results. It uses an innovative method to enable background signals to be extremely low, eliminating the 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 levels of 5-methylcytosine. The kit has the following advantages and features:
- Fluorometric assay with easy-to-follow steps for convenience and speed. The entire procedure can be finished within 4 hours.
- Innovative kit composition enables background signals to be extremely low, which eliminates the need for plate blocking and allows the assay to be simple, accurate, reliable, and consistent.
- High sensitivity, of which the detection limit can be as low as 50 pg of methylated DNA and accepts as low as 20 ng of input genomic DNA
- Optimized antibody and enhancer solutions allow high specificity to 5-mC, with no cross-reactivity to unmethylated cytosine and no or negligible cross-reactivity to hydroxymethylcytosine within the indicated concentration range of the sample DNA.
- Universal positive and negative controls are included, which are suitable for quantifying methylated DNA from any species.
- Strip-well microplate format makes the assay flexible: manual or high throughput analysis.
Principle & Procedure
The MethylFlash™ Methylated DNA Quantification Kit (Fluorometric) contains all reagents necessary for the quantification of global DNA methylation. In this assay, DNA is bound to strip wells that are specifically treated to have a high DNA affinity. The methylated fraction of DNA is detected using capture and detection antibodies and then quantified fluorometrically by reading the absorbance in a fluorescence microplate spectrophotometer at excitation 530 and emission 590 nm. The amount of methylated DNA is proportional to the RFU (relative fluorescence units) measured, which can be calculated with the kit's included formulas for relative methylation status of two different DNA samples or 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 for a flexible assay in manual or high throughput analysis. Universal positive and negative controls are included with the kit for quantifying methylated DNA from any species such as mammals, plants, fungi, bacteria, and viruses.
Safe and Convenient
All the needed reagents, including negative controls and positive controls, for quantification of global DNA methylation are conveniently packaged in the kit. The direct fluorometric quantification of DNA samples replaces obsolete 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 for accurate quantification of methylated DNA to be achieved with high sensitivity and specificity. The detection limit of the input DNA can be as low as 50 pg of methylated DNA.
Comparison of Available ELISA-based Global DNA Methylation Assay Methods
| ||MethylFlash (Fluorometric)||Competitor Z||Competitor C|
||Excellent detection limit: 0.05 ng of 5-mC DNA
||Good detection limit: >0.5 ng of 5-mC DNA
||Very poor detection limit: >1000 ng of 5-mC DNA
||>20 with very low background
|| <4 with high background
||No need for plate blocking and denaturation of DNA
||Requires plate blocking and DNA denaturation
||Requires plate blocking, DNA digestion, and antibody competitive incubation in an additional plate
|| <4 hr
|| <4 hr
|| >8 hr
|DNA Type & Species
||Universal for any species, both ssDNA and dsDNA
||Human and mouse ssDNA only
||Specific to 5-mC only
||Cross-reaction to 5-hmC
||Both relative and absolute quantification
||Stable, quantitative in absolute amount of 5-mC, and can be universally used for any species
||Unstable, cannot be quantitative in amount, and can only be used for human or mouse
|Minimum Input Amount
|Accuracy of Detection
||High, stable and quantified standard control. Proven close correlation with LC-MS analysis by users
||Unclear, unstable and un-quantified standard control
|% 5-mC calculation
||Very high published citation count
||Low published citation count
||Low published citation count
* Insufficient information provided by datasheet or product failed to produce data to make conclusive determination
Fig. 1. Schematic procedure for the MethylFlash™ Methylated DNA Quantification Kit (Fluorometric).
Fig. 2. Demonstration of high sensitivity and specificity of methylated DNA detection achieved by the MethylFlash™ kit. Synthetic unmethylated DNA (contains 50% of cytosine) and methylated DNA (contains 50% of 5-methylcytosine) were added into the assay wells at different concentrations and then measured with the MethylFlash™ Methylated DNA Quantification Kit (Fluorometric).