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Global 5-hmC DNA ELISA Kit

detect and quantify 5-hmC levels in genomic DNA

 

Global 5-hmC DNA ELISA Kit Overview

Glbal 5-hmC Kit
 

The Global 5-hmC DNA ELISA Kit is designed to detect and quantify 5-hydroxymethylcytosine (5-hmC) levels in genomic DNA from samples with different disease states, treatment conditions, clinical outcomes or environmental backgrounds. Quantification of 5-hmC using the Global 5-hmC DNA ELISA Kit is consistent with mass spectrometry, while allowing for higher throughput processing of samples and requiring only nanogram amounts of genomic DNA.

DNA hydroxymethylation has been shown to play an important role in both embryonic and neuronal development and disease pathogenesis. Research indicates a global decrease in 5-hmC levels associated with numerous cancers, while changes to the abundance of 5-hmC has been associated with neurodegenerative disorders making it a proposed molecular marker and therapeutic target.

The Global 5-hmC DNA ELISA Kit contains an optimized protocol and all necessary reagents to perform DNA fragmentation, plate coating, capture, and colorimetric detection of 5-hmC. For quantitative analysis, a synthetic DNA standard oligo containing an exact number of 5-hmC is included in the kit. Serial dilutions of the standard can be utilized to generate a standard curve for determination of both molar and % 5-hmC values in the DNA samples.

Global 5-hmC DNA ELISA Highlights

Flow Chart of Global 5-hmC DNA ELISA Kit
Flow Chart of Global 5-hmC DNA ELISA Kit
(Click image to enlarge)

 

Global 5-hmC DNA ELISA Kit Contents & Storage

The Global 5-hmC DNA ELISA Kit contains sufficient reagents to perform 96 reactions. While the kit is shipped on dry ice, it 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
  • MseI Reaction Buffer (10X); Store at -20°C
  • 5-hmC DNA Standard (20X, 100 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
  • Buffer W2 (2X); Store at 4°C
  • 96-well plate; Store at 4°C
  • Stop Solution; Store at RT
  • Plate sealer; Store at RT
  • 0.2 ml PCR stripwell tubes; Store at RT
 

Global 5-hmC DNA ELISA Kit Data

Figure 1: Synthetic 5-hmC DNA standard generates reliable and consistent standard curves.

Raw absorbance (450 nm) was plotted versus ng of the 5-hmC DNA standard in each well. 50 ul of each dilution of the 5-hmC DNA standard was added to each well. Development time was 7 minutes. The best curve fit was determined using Excel linear regression.

Figure 2: Global 5-hmC DNA ELISA Kit quantification of 5-hmC is nearly identical to mass spectrometry.

The Global 5-hmC DNA ELISA Kit was used to determine the % 5-hmC in genomic DNA isolated from human tissues. For each assay 10 ng/well of genomic DNA was used. The % 5-hmC was calculated for each sample using a standard curve generated from a serial dilution of the included 5-hmC DNA Standard. Development time was 22 minutes. The data was compared to mass spectrometry data obtained from 500 ng of the same DNA. Results show the Global 5-hmC DNA ELISA Kit provides equivalent quantification to mass spectrometry, while requiring only a fraction of the starting material.

Figure 3: 5-hmC quantification using the Global 5-hmC DNA ELISA Kt is superior to competitor’s kit.

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

 

Global 5-hmC DNA ELISA Kit Documents

 



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