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Global 5-hmC Quantification Kit

Quantify global changes in hydroxymethylcytosine levels

Active Motif's Global 5-hmC Quantification Kit is an ELISA-based assay designed to detect and quantify 5-hydroxymethylcytosine (5-hmC) levels in genomic DNA from different sample types (e.g. normal and diseased), treatment conditions, clinical outcomes or environmental backgrounds. The Global 5-hmC Quantification Kit enables higher throughput processing of samples, requires only nanogram amounts of genomic DNA per well and provides results that are consistent with mass spectrometry quantification.

Assay results for Active Motif's Global 5-hmC Quantification Kit.
Assay results for Active Motif's Global 5-hmC Quantification Kit.

(Click image to enlarge)

Global 5-hmC Quantification Advantages

  • Highly sensitive; can detect a little as 0.02% 5-hmC
  • Suitable for use with tissue-derived genomic DNA
  • Can detect 5-hmC from as little as 10-50 ng of input DNA
  • Includes DNA standards for quantification of global 5-hmC levels
  • Higher throughput quantification than mass spectrometry

Hydroxymethylation is known to play a role in transcriptional regulation and embryonic development. Research has indicated it may also serve as a prognostic indicator in certain cancers and neurodegenerative disorders. Because gene-specific analysis techniques do not provide information about the global levels of DNA hydroxymethylation within a genome and mass spectrometry methods require large amounts of DNA and provide low throughput analysis, Active Motif developed the Global 5-hmC Quantification Kit. It is a simple, ELISA-based assay to detect and quantify global changes in 5-hydroxymethylcytosine levels. The colorimetric readout is easily quantified using a microplate reader at 450nm.

Active Motif’s Global 5-hmC Quantification Kit contains an optimized protocol and all necessary reagents to perform DNA fragmentation, plate coating, capture, and colorimetric detection of 5-hmC. For added convenience, DNA standards containing known levels of 5-hmC are included in the kit. Standards can be utilized to generate a standard curve for determination of % 5-hmC values for the DNA samples.

To learn more about the Global 5-hmC Quantification Kit, click on the Method, Data, or Contents tabs below. To view a manual or other related documents, click on the Documents tab below.

 
Name Format Cat No. Price  
Global 5-hmC Quantification Kit 1 x 96 rxns 55018 $405 Buy Now

How does the Global 5-hmC Quantification Kit work?

Diagram demonstrating the method of the Global 5-hmC Quantification Kit from Active Motif
Figure 1: Schematic of the Global 5-hmC Quantification Kit.

In the Global 5-hmC Quantification Kit, the genomic DNA of interest is first enzymatically digested using the provided MseI enzyme and heat denatured before addition to coated 96-well plates. Unbound DNA fragments are then washed away. A 5-hydroxymethylcytosine antibody and a secondary antibody conjugated to horseradish peroxidase (HRP) are used for detection of the hydroxymethylated fragments. The colorimetric readout is then easily quantified by spectrophotometry on a microplate reader. Results are compared to the included DNA standards to extrapolate the percent 5-hmC in the DNA sample.

Global 5-hmC Quantification Kit

The Global 5-hmC Quantification Kit is an ELISA-based assay that is designed for detection and quantitation of 5-hydroxymethylcytosine (5-hmC) levels of tissue-derived genomic DNA. DNA standards with known amounts of 5-hmC are provided for generating a standard curve to extrapolate the percentage of 5-hydroxymethylcytosine in the DNA samples.

Comparison of Active Motif's Global 5-hmC Quantification Kit and Mass Spectrometry
Figure 1: Comparison of 5-hmC quantification using the Global 5-hmC Quantification Kit and mass spectrometry.

The Global 5-hmC Quantification Kit was used to determine the % 5-hmC in genomic DNA isolated from mouse and human tissues. Genomic DNA was tested in the range of 20 ng/well (human brain) up to 50 ng/well (all other DNA samples). The % 5-hmC was calculated for each sample using the included DNA standards. The data was compared to mass spectrometry data obtained from 500 ng of the same DNA samples. Results show the Global 5-hmC Quantification Kit provides equivalent quantification, while requiring only a fraction of the starting material.

 

Global 5-hmC Quantification standard curve
Figure 2: Graphical representation of the standard curve generated with the Global 5-hmC Quantification Kit.

The % 5-hydroxymethylcytosine (% 5-hmC) represented by each DNA standard was plotted on the x-axis and the Net OD 450nm readings for the standards were plotted on the y-axis. The best curve fit was determined using Excel Linear Regression. The calculated % 5-hmC values for the DNA samples were extrapolated from the curve using the trendline equation.

 

Comparison of 5-hmC Quantification Methods
Figure 3: Comparison of various 5-hmC quantification methods.

Genomic DNA samples from human and mouse tissues were analyzed for % 5-hydroxymethylcytosine using either mass spectrometry, Active Motif's Global 5-hmC Quantification Kit or Competitor Z's Global 5-hmC Kit. Each method was performed as recommended by the manufacturer. The % 5-hmC was then plotted for comparison of the various methods. Active Motif's Global 5-hmC Quantification Kit produced results most similar to mass spectrometry analysis, while Competitor Z's quantification showed significant differences from the mass spectrometry values.

Contents & Storage

The Global 5-hmC Quantification Kit contains sufficient reagents to perform 96 reactions.

The Global 5-hmC Quantification Kit is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please confirm receipt of all reagents upon arrival and store items at the appropriate temperatures as indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. This kit includes the following components:

  • MseI enzyme (10 U/μl); Store at -20°C
  • 10X MseI Reaction Buffer; Store at -20°C
  • 5-hmC DNA Standards A, B, C, D, E, F & G (10 ng/μl); Store at -20°C
  • DNA Binding Agent (lyophilized); Store at -20°C
  • 5-Hydroxymethylcytosine pAb; Store at -20°C
  • Anti-rabbit HRP-conj. secondary Ab; Store at 4°C
  • Antibody Dilution Buffer; Store at 4°C
  • 20X Wash Buffer; Store at 4°C
  • Developing Solution; Store at 4°C
  • 96-well plate; Store at 4°C
  • Stop Solution; Store at RT
  • 10X Sample Dilution Buffer; Store at RT
  • 10X Buffer W; Store at RT
  • Plate sealer; Store at RT
  • 0.2 ml PCR stripwell tubes; Store at RT