How it Works

The EpiTect Methyl II PCR Array System relies on the differential cleavage of target sequences by two different restriction endonucleases whose activities require either the presence or absence of methylated cytosines in their respective recognition sequences. As real-time PCR quantifies the relative amount of DNA remaining after each enzyme digestion, the methylation status of individual genes and the methylation profile across a gene panel are reliably and easily calculated. The high yield of DNA from the restriction digests and PCR amplification allow the analysis of smaller, more heterogeneous samples.

Download User Manual  Tour the FREE Data Analysis Template

What It Offers:

• Guaranteed Performance - Ready-to-use for DNA methylation analysis

• Time & Cost Savings - Less than 30 min hands-on time to analyze up to 94 genes

• Easy Data Analysis - FREE Excel-based data analysis template available

You can easily perform an EpiTect Methyl II experiment in your own laboratory using any 96-well or 384-well real-time PCR instrument that you have access to.
Or you can send your DNA samples to us and take advantage of our PCR Array Services.

Array Layout:
The PCR Arrays are available in both 96- and 384-well plate formats to analyze the methylation status of 22 or 94 genes related to a disease state, such as specific types of cancer, or development pathways related to specific cells or tissues. The Signature panels of 22 genes are arranged on either 96-well or 384-well plates to simultaneously characterize all four restriction digests from either one or four different DNA samples, respectively. The more Complete panels of 94 genes are arranged on 96- or 384-well plates to characterize all four restriction digests from one DNA sample either on four plates or all on the same plate, respectively.

For more detail on PCR Array layout, see the "Gene Table" link for the individual array products.

Data Interpretation
EpiTect Methyl II PCR Arrays provide gene methylation status as percentage unmethylated (UM) and percentage methylated (M) fraction of input DNA. "UM" represents the fraction of input genomic DNA containing no methylated CpG sites in the amplified region of a gene. "M" represents the fraction of input genomic DNA containing two or more methylated CpG sites in the targeted region of a gene. The number of CpG sites methylated in a targeted region can vary within the fraction of methylated input DNA.

In the figure above, each horizontal bar represents the targeted region of a gene from one genome. Biological samples usually contain many genomes derived from many cell types. For simplicity, five such genomes are depicted here. Light and dark circles represent unmethylated and methylated CpG sites, respectively.

Performance Data

To verify the reliability of the EpiTect Methyl II PCR Array System, its results and sensitivity were compared with bisulfite Sanger sequencing, the gold standard in DNA methylation analysis

Same Results as Bisulfite Sanger Sequencing

To validate the reliability of the system, EpiTect Methyl II PCR system results (EpiTect Methyl II PCR) were compared with bisulfite Sanger sequencing, the gold standard in DNA methylation analysis. The methylation status of the cadherin 13 (CDH13) gene promoter was analyzed using either bisulfite sequencing or EpiTect Methyl II PCR Assays in two different cell lines. EpiTect Methyl II PCR Assays revealed that 100% of total input DNA had methylated CDH13 gene promoter in MB231 cell lines whereas 100% of input DNA from HeLa cell line had unmethylated CDH13 promoter. Importantly, EpiTect Methyl II PCR Assays yielded results similar to Bisulfite Sequencing.

Same Sensitivity as Bisulfite Sanger Sequencing

Primary tumors are typically very heterogeneous, containing a mixture of both cancerous and noncancerous cells. Therefore, reliable tumor characterization requires detecting smaller amounts of methylated DNA diluted in an unmethylated background. To test the sensitivity of EpiTect Methyl II PCR system to detect methylated DNA diluted in an unmethylated background, SKBR3 breast cancer cell line and normal blood genomic DNA (encoding methylated and unmethylated HIC1, respectively) were mixed in different ratios. EpiTect Methyl II Human HIC1 PCR Primers Assays were used to detect the methylation status of the mixed sample. Result show that the percentage of methylated HIC1 relative to total promoter DNA in each mixture was detectable even down to six percent of the total DNA sample showing the high sensitivity of EpiTect Methyl II PCR system.

Application Data

Gene promoter methylation is the most common epigenetic mechanism silencing tumor suppressor genes during oncogenesis. Almost all cancer-related signaling pathways are affected by methylation, and the number of genes affected in each major type of cancer is still rapidly growing. However, even the most relevant genes have not yet been correlated to individual cancer types or subtypes in order to better define biological pathways and mechanisms leading to oncogenesis and in order to properly develop DNA methylation biomarkers. The EpiTect Methyl II PCR System provides an ideal reagent for such studies, without bisulfite conversion of DNA. The following experiments demonstrate that EpiTect Methyl II PCR Arrays can both verify known and discover new DNA methylation cancer biomarkers

EpiTect Methyl II PCR Arrays are used to screen Breast Cancer Gene Methylation Status in Breast Cancer Cell Lines.The heat map compares the methylation status of 22 genes in the genomic DNA of three breast cancer cell lines and blood genomic DNA (used as an unmethylated control) determined with the Human Breast Cancer EpiTect Methyl II Signature PCR Arrays. The results further strengthen the correlation of these biomarkers with breast cancer.

Biomarker / Pathway Discovery
Cancer progresses through aberrant cell differentiation due to alterations in gene expression. Transcription factors regulate gene expression, and many tumor suppressor genes and oncogenes, defined by classical genetic methods, encode transcription factors. Does the methylation status of transcription factor genes differ between cancer and normal cells?

EpiTect Methyl II DNA Methylation PCR Arrays Discover New Candidate Breast Cancer DNA Methylation Biomarkers. The heat map compares the methylation status of a panel of 79 transcription factor genes in six different breast cancer cell lines (some in duplicate) and a normal epithelial cell line as determined using 384-well EpiTect Methyl II Custom PCR Arrays. These breast cancer cell lines also methylate this gene panel potentially providing a new source of cancer biomarkers.

These results are consistent with the notions that aberrant expression of transcription factors controlling cell differentiation plays key roles in oncogenesis and that transcription factors can be tumor suppressors.