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. 2. Sensitive detection of gene-specific hydroxymethylation by hMeDIP-QPCR | Human brain DNA (500 ng) was fragmented to 200-600 bps with EpiSonic 1000 (Cat. No. EQC-1000). 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 Cts.
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.