Comparison of Available ELISA-based Global DNA Methylation Assays
An ELISA-based global DNA methylation assay can be one of the most successful assays for researchers looking to efficiently measure total 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), or 5-formylcytosine (5-fC) in their samples. However, not all commercially available ELISA-like global DNA methylation kits are created equal. With several commercial kits offered, it can be difficult to pinpoint the exact capabilities and performance abilities of each kit to ensure successful quantification of total 5-mC and total 5-hmC.
Global DNA methylation kits on the market can vary greatly in terms of input requirements, sensitivity, protocol time, and effectiveness, even though they may all utilize an ELISA-based method. It is crucial to have a comprehensive overview of what these kits provide such as high-throughput, high sensitivity, and low input capabilities, in order to set one’s global DNA methylation research up for success. Understanding the capabilities of each global DNA methylation assay kit in regard to accurate measurement of total 5-hmC and total 5-mC can help investigators optimize their epigenetic research for results that are of publication quality.
Below are two tables comparing popular ELISA-based global DNA methylation kits that are currently available to researchers. Also see how to overcome the limitations of the important global DNA methylation assay.
Comparison of Global 5-mC Quantification Kits
| METHYLFLASH | KIT Z | KIT C | |
|---|---|---|---|
| Assay Principle | Direct ELISA | Direct ELISA | Indirect ELISA |
| Assay Format | 96-well plate | 96-well plate | 96-well plate |
| Sensitivity | Excellent detection limit: 0.1 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* |
| Procedure 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 | 20 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
Comparison of Global 5-hmC Quantification Kits
| METHYLFLASH | KIT Z | |
|---|---|---|
| Assay Principle | Direct ELISA – Detection antibody specifically and directly detects 5-hmC | Indirect ELISA – Detection antibody detects dsDNA (detected signals come from the intact DNA strand and are only dependent on the amount of intact DNA, not actually from 5-hmC and are not dependent on the amount of 5-hmC) |
| Assay Format | 96-well plate | 96-well plate |
| Sensitivity | Excellent detection limit of 0.04 ng of 5-hmC DNA | Unclear, as the detected signals are from intact DNA strands |
| S/N Ratio | > 20 with very low background | < 4 with high background |
| Procedural Convenience | No need for plate blocking and denaturation of DNA | Requires plate blocking and DNA denaturation |
| Protocol Time | < 4 hr | < 5 hr |
| DNA Type & Species | Universal for any species, both ssDNA and dsDNA | Only ssDNA from limited number of species |
| Specificity | High, the antibody only detects 5-mC | Unclear, as the assay principle is not reasonable |
| Quantification Type | Both relative and absolute quantification | Relative only |
| Standard Control | Stable, quantitative in absolute amount of 5-mC and can be universally used for any species | Unclear |
| Minimum Input Amount | 20 ng | 100 ng |
| Accuracy of Detection | Stable and quantified standard control with proven close correlation with HPLC-MS analysis by users | Unclear, based on biased assay principle |
| % 5-mC Calculation | Simple | Complicated |
| Patented Method | Yes | No |
| Popularity | Very high published citation count | Low published citation count |
| Support Expertise | Since 2006 | Since 2012 |