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EpiQuik Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit

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For antibody-based capture of hydroxymethylated DNA fragments for microarray, PCR, or NGS applications

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Suggested Workflow
DNA Isolation
 
 
DNA Shearing
 
 
MeDIP
 
 
PCR Analysis
 
Schematic procedure of the EpiQuik™ Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit.
Sensitive detection of gene-specific hydroxymethylation by hMeDIP-PCR | Human brain DNA (500 ng) was fragmented to 200-600 bps. The fragmented DNA was used for hydroxymethylated DNA enrichment with the EpiQuik™ hMeDIP Kit. Eluted DNA was analyzed by real time PCR with primers specifically for OCT4 or GAPDH sequences in the promoter regions. Results show that the promoter region is hydroxymethylated in OCT4 but not in GAPDH. Fold-enrichment represents the amount of recovered DNA and was calculated based on the Ct Value. Fig. 3. Selective enrichment of hydroxymethyated DNA with the EpiQuik™ hMeDIP Kit | 50 pg of unmethylated, methylated, and hydroxymethylated DNA control were each spiked into fragmented human genomic DNA (500 ng). hMeDIP was processed with the 5-hmC antibody and non-immune IgG included in the kit. Eluted DNA was analyzed by real time PCR with the control primers included in the kit to detect the presence of spiked control DNA. Fold-enrichment represents the amount of recovered control DNA and was calculated based on the Cts.
Input Type: DNA
Research Area: DNA Methylation
Target Application: Immunoprecipitation
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-1038-2424 reactions $338.00 
P-1038-4848 reactions $599.00 
P-1038-9696 reactions $1,059.00 
Availability: Usually Ships In 1-2 Days 
Product Overview

The EpiQuik™ Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit is a complete set of optimized buffers and reagents to carry out a successful hMeDIP procedure for the selective enrichment of DNA fragments containing 5-hydroxymethylcytosine (5-hmC) in a high throughput format using DNA isolated from any species. The hydroxymethylated DNA that is enriched with this kit can be used in various downstream applications including PCR (hMeDIP-PCR) and microarrays (hMeDIP-chip) for investigating gene-specific hydroxymethylation in cells or tissues.

About 5-Hydroxymethylcytosine
5-hmC is a modified form of cytosine, recently discovered in animal tissues, and plays an important and different role than 5-mC in regulation of DNA methylation, chromatin remodeling, and gene expression, particularly in brain-specific gene regulation. Quite recently, 5-hmC was observed to be linked to epigenetic reprogramming in mammalian zygotes. However, the exact functions of 5-hmC have not yet been fully identified since gene-specific distribution of 5-hmC is unknown due to the inability of currently used DNA methylation analysis methods to distinguish between 5-hmC and 5-mC.

Product Features
Because of the presence of 5-hmC in DNA with unclear functions in gene regulation and because of the discovery of enzymes that produce 5-hmC, it is crucial to identify hydroxymethylation status in specific gene loci, which would help to better understand methylation-based epigenetic regulation of gene functions. To achieve this, Epigentek has developed an innovative method to capture DNA fragments containing 5-hmC, and incorporated this method into its EpiQuik™ Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit. This kit uses a high affinity 5-hmC antibody to selectively capture double-stranded or single stranded DNA fragments containing 5-hmC. The kit has the following features:

  • Extremely fast and convenient protocol with a total procedure time (from input sample to ready-to-use hydroxymethylated DNA) of less than 3 hours, which includes a minimal handling time of less than 20 minutes.
  • Flexible 96 stripwell microplate format makes the assay very easy to handle: manual method with one reaction at a time or high throughput method with 96 reactions at a time.
  • Highly efficient enrichment ratio of positive/negative control > 1000.
  • Low DNA input requirement of as low as 0.1 µg per reaction.
  • High reproducibility through pre-optimized hMeDIP conditions.
  • Compatible with various downstream analysis workflows including hMeDIP-PCR and hMeDIP-chip.

Principle and Procedure
The EpiQuik™ Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit contains all reagents required for carrying out a successful hMeDIP procedure using DNA isolated from mammalian cells or tissues. This kit includes a positive control DNA fragment, a negative control non-immune IgG, and control primers that can be used with the positive control to demonstrate the enrichment efficacy for hydroxymethylated DNA with the kit reagents and protocol. The positive control DNA containing 5-hmC can be immunoprecipitated by a 5-hmC antibody but not by a non-immune IgG. In this hMeDIP, immunoprecipitation of 5-hmC-enriched DNA fragments is processed in a microplate under optimized reaction conditions, which enables hMeDIP to be completed within 3 hours with high efficiency. Immunoprecipitated hydroxymethylated DNA is then cleaned, released, and eluted. Eluted DNA can be used for various downstream applications including PCR (hMeDIP-PCR) and microarray (hMeDIP-chip).

Easy, Fast, and Flexible
The entire hMeDIP procedure has easy-to-follow steps for convenience and speed, which can be completed in 3 hours from DNA shearing to hMeDNA elution. The 96 stripwell microplate format permits manual handling by doing 1 reaction at a time or high throughput handling by doing 96 reactions at a time. Hydroxymethylated DNA fragments and non-immune IgG are included with the kit as the positive control and negative control, respectively, for hydroxymethylated DNA enrichment from any species including mammalian, plant, fungal, bacterial, and viral types.

Selective and High Enrichment
The unique procedure and proprietary kit compositions allow for a selective and high enrichment of hydroxymethylated DNA from genomic DNA samples for gene-specific hydroxymethylation analysis. Combining real time PCR with this kit, 5-hmC enrichment in the OCT4 promoter region was detected to be 110 times higher than that in the GAPDH promoter region (see Fig. 2), indicating that the OCT4 promoter is hydroxymethylated. There is no cross-reactivity to methylcytosine and unmethylated cytosine so that only hydroxymethylated DNA (5-hmC) is enriched (see Fig. 3).

Compatible With MeDIP
The EpiQuik™ hMeDIP kit can be used with MeDIP procedures for a simultaneous analysis of hydroxymethylated and methylated statuses of specific gene loci.

Responsive, Reliable, and Practical
Based on its working principle and the microplate format, the kit can be used in a high throughput manner with DNA from any species in a variety of forms including cultured cells, fresh and frozen tissues, paraffin-embedded tissues, blood samples, and body fluid samples. To demonstrate the capabilities of the kit, it has been successfully used for capturing hydroxymethylated DNA fraction from genomic DNA isolated from human brain tissues. The percent content of hydroxymethylated DNA in human brains enriched with the kit (0.80%) is comparable to that measured with the EpiQuik™ Hydroxymethylated DNA Quantification Kit (0.67%), suggesting that the hydroxymethylated DNA fraction is fully captured with the hMeDIP kit.


Fig. 1. Schematic procedure of the EpiQuik™ Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit.

Fig. 2. Sensitive detection of gene-specific hydroxymethylation by hMeDIP-PCR | Human brain DNA (500 ng) was fragmented to 200-600 bps. The fragmented DNA was used for hydroxymethylated DNA enrichment with the EpiQuik™ hMeDIP Kit. Eluted DNA was analyzed by real time PCR with primers specifically for OCT4 or GAPDH sequences in the promoter regions. Results show that the promoter region is hydroxymethylated in OCT4 but not in GAPDH. Fold-enrichment represents the amount of recovered DNA and was calculated based on the Ct Value. 

 
Fig. 3. Selective enrichment of hydroxymethyated DNA with the EpiQuik™ hMeDIP Kit | 50 pg of unmethylated, methylated, and hydroxymethylated DNA control were each spiked into fragmented human genomic DNA (500 ng). hMeDIP was processed with the 5-hmC antibody and non-immune IgG included in the kit. Eluted DNA was analyzed by real time PCR with the control primers included in the kit to detect the presence of spiked control DNA. Fold-enrichment represents the amount of recovered control DNA and was calculated based on the Cts.

  EpiQuik™ hMeDIP Supplier 1's hMeDIP Supplier 2's hMeDIP
Working Principle Immunocapture Immunocapture Immunocapture
Working Format Microplate-based Beads-based Beads-based
Max Number of Reactions Per Run 96 10 16
High Throughput Capable Yes No No
Convenience (Assay Steps) 12 steps 20 steps 28 steps
Total Procedure Time < 3 hours ~24 hours ~24 hours
Physical Handling Time < 15 minutes > 1 hour > 1 hour
Amount of Input DNA 0.1-1 µg ss/dsDNA 0.1-1 µg ss/dsDNA 1.2 µg ss/dsDNA
Fold Enrichment With Spiked 5-hmC Control >1000 >1000 >1000
Fold Enrichment With Genomic DNA Samples From Cells/Tissues >100 N/A N/A
Yield of Enriched hmeDNA from Genomic DNA Samples ~4 ng per 500 ng of human brain DNA N/A N/A
Specificity (Positive/Negative Ratio) >99% >99% >99%
Cost Per Reaction $10 $35 $47

Product Components

Wash Buffer
Antibody Buffer
hMeDIP Solution
DNA Release Buffer
Non-Immune IgG (0.6 mg/ml)
5-hmC Antibody (0.6 mg/ml)
Control DNA (500 ng/ml)
Proteinase K (10 mg/ml)
Control Primer-Forward (20 µM)
Control Primer-Reverse (20 µM)
8-Well Assay Strips (With Frame)
Adhesive 8-Well Strip Film
User Guide

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 info@epigentek.com along with your contact information and institution name.

 

Customer Reviews

Rating by w****@jhsph.edu Verified Purchase Reviewed on: Tuesday 30 September, 2014
Application Description
Attached the results on the 5hmC enrichment PCR from experiment using your kit and ZymoResearch Kit (we are currently using). In general, I’m happy with the results. We probably want to use the voucher for getting the 96 reaction kit. We would also like to order the TET activity Kits and Nuclear Extraction Kits.

Procedural Details
Objective: Measure the 5hmC enrichment of GOI promoter after drug treatment
Principle of 5hmC enrichment (Epigentek): Antibody based, immunoprecipitation (5hmC/IgG Ab)
Principle of 5hmC enrichment (Zymo Research): Chemical Modification (glucosylation) followed by Immunoprecipitation (JBP-1 binding
Sample Species: Human Airway Smooth Muscle Cells
Sample amount (1000ug genomic DNA followed by MseI fragmentation and separate into 2 groups IgG (or Mock) and 5hmC ( or JBP))
* take 1:100 fragmentated DNA for input
Detection method (real-time PCR on ABI7500Fast using SsoFast SYBR-Green from BIORAD, same PCR plate)

Other Thoughts
Pro and Con ( for Epigenek):
PRO:
1) shorter processing time (4hr vs 6h)
2) less procedures
3) increased sensitivity for abundant genes (Gene 2 and 3 only, Ct<30) showing lower Ct value (2-4 Ct differences). Not Gene 1 (Ct>35)
CON:
1) Need a heat block for 65C (cannot perform in PCR machine, less convenient)


Comparing the results using 5MeDIP PCR enrichment kit (Epigentek) and 5hmC enrichment PCR kit (Zymo Research).
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