ELISA methods 5-hmC quantificationA popular method for measuring 5-hydroxymethylcytosine (5-hmC) in samples is an ELISA-based technique. Choosing the appropriate commercial kit that will provide both high sensitivity and specificity is crucial for a successful DNA hydroxymethylation study, especially avoiding cross-reactivity to methylcytosine and unmethylated cytosine. The following table highlights the benefits of Epigentek’s MethylFlash Hydroxymethylated DNA Quantification Kit for measuring global 5-hmC, including an impressively low input requirement, high sensitivity and specificity, and universal species compatibility – and compares it to alternative available ELISA-based Global 5-hmC DNA Hydroxymethylation assay methods:


MethylFlash Competitor Z
Assay Principle Direct ELISA – Detection antibody specifically and directly detects 5-hmC Indirect ELISA – Detection antibody detects dsDNA, (detected signals is dependent only on intact DNA strands and does not quantify actual total 5-hmC)
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