Differential Methylation Analysis
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At the PMGC we provide both microarray and high throughput sequencing solutions for methylation analysis.

Differential methylation hybridization (DMH) is a microarray-based technique used to analyze DNA methylation on a global scale. This method was first described by Huang et al. (Hum Mol Genet, 1999, 8:459-470). It allows for the identification of changes in DNA methylation patterns that is commonly observed in cancer and other disease states.

Agilent offers human and mouse (CpG island and promoter) arrays for DMH experiments, as well as custom designed arrays for any species. With the Agilent platform, two methods can be used to examine DMH: MeDIP, an immunocapturing approach to enrich methylated DNA, and a method that involves methylation sensitive restriction enzymes and PCR amplification.

With Illumina methylation assays, researchers can quantitatively interrogate methylation sites at single-nucleotide resolution. The Illumina Infinium HD Methylation Array accomplishes this high multiplexing by combining bisulfite conversion of genomic DNA and whole-genome amplification (WGA) sample preparation with direct, array-based capture, and enzymatic scoring of the CpG loci.

To assess the methylation status using the Illumina HiSeq there are multiple methods available. One is to combine sequencing with bisulphite conversion of DNA (whole genome bisulphite sequencing); however, this can be extremely costly and time consuming for large studies. Alternatively, MeDIP-Seq or Reduced representation bisulfite sequencing (RRBS), where the genome is first digested with a restriction enzyme for CpG recognition sites, however, both these methods tend to be biased towards repeat sequences and CpG-rich sequences. There are also some kits available such as Agilent's SureSelect Methyl-Seq.

For the methylation analysis experiment services, we ask that researchers prepare the genomic DNA and perform methylation-sensitive restrictions and PCR amplification, or perform MeDIP (for Agilent platform). Similarly, researchers are required to provide bisulfite converted genomic DNA for the Illumina platform (microarray or sequencing).

Differential methylation experiments can be performed using the Illumina sequencing or Illumina array platforms. Not sure which platform is best suited to your experimental needs? Click here.


Quick Questions:

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What kind of samples should be submitted and how much is required?

For the Agilent microarray platform a minimum of 6 μg of each PCR amplified genomic DNA sample per array is required. As well, we ask that you submit an additional 1μg of each PCR amplified genomic DNA sample for analysis on an agarose gel and, if necessary, for OD purposes.

We ask that users of the DMH service complete up to, and including, step 2.2.4 of the Yan et al. protocol. If you choose another protocol, we still ask that you send PCR amplified genomic DNA that has been column purified. Users of the service should also complete the appropriate checklist and submit the information with the samples. Please contact us for more information.

For Illumina arrays 500ng of bisulfite-converted genomic DNA is required using ZYMO EZ DNA methylation kit (Zymo Research).

For Methyl-Seq: RBBS method requires 5 μg of input DNA, whole-genome bisulphite sequencing method requires 10 μg of input DNA.

Please normalise concentration of all DNA samples submitted.

Why use the DMH technique with CpG island microarrays?

Methylation of CpG islands located in promoter regions regulates the transcription of downstream genes. It has been shown that DMH using CpG island arrays is useful in classifying tumours according to their methylation profiles.

What is used as a control for a DMH experiment?

Typically, a sample that has been treated with a methylation sensitive enzyme is co-hybridized with a sample that has not been treated with a methylation sensitive enzyme or a methylation insensitive enzyme.