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MethylFlash Methylated DNA 5-mC Quantification Kit (Colorimetric)


For absorbance-based quantitation of global DNA methylation in an ELISA-like format

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Suggested Workflow
DNA Isolation
DNA Methylation Quantification
Schematic procedure for the MethylFlash™ Methylated DNA Quantification Kit (Colorimetric).
Demonstration of high sensitivity and specificity of methylated DNA (5mC) 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 (Colorimetric).
5-mC content levels in various sample types measured by MethylFlash™ technology versus HPLC-MS/MS, demonstrating a reliable correlation between the two techniques.
Input Type: DNA
Research Area: DNA Methylation
Target Application: Amount Quantitation
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-1034-4848 assays $299.00 
P-1034-9696 assays $539.00 
Availability: Usually Ships in 1 Day or Same day delivery Same Day NY Delivery 
Product Overview

The MethylFlash™ Methylated DNA Quantification Kit (Colorimetric) is a complete set of optimized buffers and reagents to colorimetrically quantify global DNA methylation by specifically measuring levels of 5-methylcytosine (5-mC) in an ELISA-like 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 (Colorimetric) for simultaneously quantifying both methylated DNA and hydroxymethylated DNA.

Product Features
The MethylFlash™ Methylated DNA Quantification Kit (Colorimetric) is a further refinement of our previous Methylamp™ 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:

  • Colorimetric 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 0.2 ng of methylated DNA and accepts as low as 50 ng of input genomic DNA.
  • Optimized antibody and enhancer solutions allow high specificity to 5mC, 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 (Colorimetric) 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 through an ELISA-like reaction by reading the absorbance in a microplate spectrophotometer at 450 nm. The amount of methylated DNA is proportional to the OD intensity 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 (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 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 colorimetric 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 0.2 ng of methylated DNA.

Comparison of Available ELISA-based Global DNA Methylation Assay Methods 

 MethylFlash(Colorimetric)Competitor ZCompetitor C
Assay Principle Direct ELISA Direct ELISA Indirect ELISA
Format 96-well plate 96-well plate 96-well plate
Sensitivity Excellent detection limit: 0.2 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
S/N Ratio >20 with very low background  <4 with high background  N/A*
Procedural Convenience 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
Protocol Time  <4 hr  <4 hr  >8 hr
DNA Type & Species Universal for any species, both ssDNA and dsDNA Human and mouse ssDNA only Various species
Specificity Specific to 5-mC only Cross-reaction to 5-hmC N/A*
Quantitation Type Both relative and absolute quantification Relative Relative
Standard Control 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 N/A*
Minimum Input Amount 50 ng 100 ng 1000 ng
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 N/A*
% 5-mC calculation Simple Complicated Not Provided
Patented Method Yes No No
Popularity Very high published citation count Low published citation count Low published citation count 
Support Expertise Since 2006 Since 2013 Since 2011

* Insufficient information provided by datasheet or product failed to produce data to make conclusive determination

Product Components

ME1 (10X Wash Buffer)
ME2 (Binding Solution)
ME3 (Negative Control, 20 µg/ml)*
ME4 (Positive Control, 20 µg/ml)*
ME5 (Capture Antibody, 1000 µg/ml*
ME6 (Detection Antibody, 400 µg/ml)*
ME7 (Enhancer Solution)*
ME8 (Developer Solution)
ME9 (Stop Solution)
8-Well Assay Strips (With Frame)
User Guide

* Spin the solution down to the bottom prior to use.

Note: The ME3 Negative Control is an unmethylated polynucleotide containing 50% of cytosine. The ME4 Positive Control is a methylated polynucleotide containing 50% of 5-methylcytosine.

User Guide & MSDS

[User Guide]*
*Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing along with your contact information and institution name.

Product Citations

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van Esterik JC et. al. (October 2014). Liver DNA methylation analysis in adult female C57BL/6JxFVB mice following perinatal exposure to bisphenol A. Toxicol Lett. 232(1):293-300.

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Wojtasik W et. al. (October 2014). Oligonucleotide treatment causes flax ß-glucanase up-regulation via changes in gene-body methylation. BMC Plant Biol. 14(1):261.

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Brown TC et. al. (September 2014). Frequent Silencing of RASSF1A via Promoter Methylation in Follicular Thyroid Hyperplasia: A Potential Early Epigenetic Susceptibility Event in Thyroid Carcinogenesis. JAMA Surg.

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Song Y et. al. (September 2014). Transgenerational impaired male fertility with an Igf2 epigenetic defect in the rat are induced by the endocrine disruptor, p,p'-DDE. Hum Reprod.

Rodríguez-Sanz H et. al. (August 2014). Early markers are present in both embryogenesis pathways from microspores and immature zygotic embryos in cork oak, Quercus suber L. BMC Plant Biol. 14(1):224.

Kim SE et. al. (August 2014). One-pot approach for examining the DNA methylation patterns using an engineered methyl-probe. Biosens Bioelectron. 58:333-7.

Naito T et. al. (August 2014). IGF2 differentially methylated region hypomethylation in relation to pathological and molecular features of serrated lesions. World J Gastroenterol. 20(29):10050-61.

Zhou Y et. al. (August 2014). Genome-Wide Demethylation by 5-aza-2'-Deoxycytidine Alters the Cell Fate of Stem/Progenitor Cells. Stem Cell Rev.

Jin L et. al. (August 2014). Genome-wide DNA methylation changes in skeletal muscle between young and middle-aged pigs. BMC Genomics. 15(1):653.

Liu Y et. al. (August 2014). Global and cyp19a1a gene specific DNA methylation in gonads of adult rare minnow Gobiocypris rarus under bisphenol A exposure. Aquat Toxicol. 156C:10-16.

Lin JR et. al. (August 2014). Vitamin C Protects Against UV Irradiation-Induced Apoptosis Through Reactivating Silenced Tumor Suppressor Genes p21 and p16 in a Tet-Dependent DNA Demethylation Manner in Human Skin Cancer Cells. Cancer Biother Radiopharm. 29(6):257-64.

El-Tantawy AA et. al. (July 2014). Changes in DNA methylation levels and nuclear distribution patterns after microspore reprogramming to embryogenesis in barley. Cytogenet Genome Res. 143(1-3):200-8.

Marino AM et. al. (July 2014). Effects of epigenetic modificators in combination with small molecule inhibitors of receptor tyrosine kinases on medulloblastoma growth. Biochem Biophys Res Commun.

Puig-Vilanova E et. al. (July 2014). Do epigenetic events take place in the vastus lateralis of patients with mild chronic obstructive pulmonary disease? PLoS One. 9(7):e102296.

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Additional Information

Customer Reviews

Rating By m******* Verified Purchase Reviewed on: Wednesday 15 March, 2017
Application Description
Given that this kit performed the same measurements as P-1030, I would recommend using the latter for global methylation quantification because P-1030 was easier (and faster) to use. Data from the P-1034 kit was of acceptable quality, but was inferior to data obtained from P-1030 because the increased number of wash steps contributed to sample contamination.

How friendly was the user guide? (1 – worst, 5 – best) 2
The user guide was straight-forward but the excessive use of abbreviations (e.g. referring to buffers or kit solutions as ME1, ME2, etc.) made it difficult to follow instructions while remembering the logic of each step. Simply changing the abbreviations to more direct, obvious ones (“wash buffer as WB” for instance) would be helpful.

How professional was the appearance and presentation of the product? (1 – worst, 5 – best) 5
The product contents were well-packaged and secured in place; all materials were present in the specified amounts. Also, there was sufficient materials to perform the total number of experiments indicated by the kit (e.g. 48 or 96) – I did not run out of wash buffer, standards, etc. which is often the case with other kits from different companies.

How would you rate the overall product? (1 – worst, 5 – best) 2
Overall, this product was easy to use one the first trial, but the instructions took too much time to interpret, and there seemed to be an excessive number of wash-steps/repetitions. Indicated incubation times also seemed very long when compared to other kits used for performing the same assay (such as P-1030).

Procedural Details
Determining differences in global DNA methylation in zebrafish (Danio rerio) embryos spawned from adult fish maintained on a vitamin E sufficient (control) vs. vitamin E deficient diet.

Global DNA methylation out of total DNA in E-sufficient (E+, blue) vs. E-deficient (E-, red-stripped) 12-day zebrafish larvae; n= 15 embryos/sample; 4 replicate samples per group; statistical analysis was performed using a student’s t-test, p<0.05. Shown are means with SEM.

Other Thoughts
Sample calculations and sample/suggested plate arrangements for sample organization were both positive attributes of this kit. The user guide contained all necessary information.

Some instructions were ambiguous and/or hard to interpret immediately due to the excessive use of non-intuitive abbreviations. Also, it would have been helpful to include certain details in different (earlier) steps – for instance, a reminder to turn on an incubator to 37C prior to beginning the assay, so that such would be ready by the time the experimenter needed to incubate the plate.

Further, the plastic wells and plate frame were flimsy and easily broke when trying to insert or remove the sample-well rows. The kit materials overall were of high quality, so making these items of higher-grade or thicker plastic would improve the product.

Data analyses with % 5-mC calculated according to the P-1034 instructions; graphs and additional statistical analyses performed used GraphPad Prism 6.0 software.
Rating By k******* Verified Purchase Reviewed on: Wednesday 11 January, 2017
Application Description
Testing the effect of chronic low dose heavy metal on global DNA methylation in MEFs

Pros: Easy to use colorimetric assay, takes less than a day, assays global DNA methylation that is accurate on a global scale versus LINE or other repetitive element DNA methylation, product arrived quickly after ordering.

Cons: There can sometimes be large variation between technical replicates. The kit recommends using technical single or duplicate samples, but I find I need to use technical triplicates.
Rating By m****** Verified Purchase Reviewed on: Monday 06 October, 2014
Application Description
We have used this kit. It was easy to use but I had some difficulty with the standards. I would rate a 4 (overall) and then a 5 (ease of use).
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