EpiTect Methyl II PCR Arrays
For application-focused DNA methylation profiling using MethylScreen technology with laboratory-verified assays
EpiTect Methyl II PCR Arrays allow the simultaneous DNA methylation of a panel of 22 or 94 gene promoters on 96- or 384-well real-time PCR plates. Genes are carefully selected based on their reported hypermethylation in a variety of experimental settings. These arrays allow correlation of CpG island methylation status with biological phenotypes or disease outcomes. Both EpiTect Methyl II Signature PCR Arrays and EpiTect Methyl II Complete PCR Arrays are available for human, mouse, or rat studies in 96- or 384-well format. EpiTect Methyl II PCR Arrays use MethylScreen technology provided under license from Orion Genomics, LLC.
EpiTect Methyl II PCR Arrays have been used to successfully verify the methylation status of tumor suppressor genes in breast cancer cell lines (see figure "Verification of breast cancer gene methylation status"). EpiTect Methyl II PCR Arrays provide high sensitivity (see figure "EpiTect Methyl II PCR Assay detects methylation in heterogeneous samples"). Results are comparable to methylation analysis using bisulfite sequencing (see figure "Comparison with bisulfite Sanger sequencing") and can provide verification for genome-wide methylation analysis studies (see figure "EpiTect Methyl II PCR Arrays generate data comparable to that from BeadChip platforms"). EpiTect Methyl II Custom PCR Arrays are also highly suited for discovery of DNA methylation biomarkers (see figure "EpiTect Methyl II PCR Arrays discover new candidate breast cancer DNA methylation biomarkers").
DNA methylation plays an important role in gene expression and it occurs almost exclusively in the context of CpG dinucleotides in the form of a covalent attachment of a methyl residue to the cytosine residue. CpG islands are regions with an elevated GC content and a high frequency of CpG dinculeotides which overlap with the promoter region of 60–70% of all human genes. Hypermethylation of CpG islands at gene promoters is mostly associated with gene silencing.
The EpiTect Methyl II PCR Array system, using MethylScreen technology, 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 (see flowchart "EpiTect Methyl II PCR Array procedure"), the methylation status of individual genes and the methylation profile across a gene panel are reliably and easily calculated (see figure "Pictorial explanation of results"). The use and analysis of both restriction digests, as well as their PCR amplification, allow the analysis of smaller, more heterogeneous samples.
The EpiTect Methyl II PCR System is an innovative technology enabling fast and accurate detection of DNA methylation status of individual genes, as well as disease- or pathway-related gene panels, without bisulfite conversion of DNA. The results of the Epitect Methyl II PCR System can then be verified using PyroMark CpG Assays. The performance of the EpiTect Methyl II PCR Arrays is guaranteed when used with the appropriate RT2 SYBR® Green qPCR Mastermix.
Array layout and format
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. Custom panels are also available. The signature panels of 22 genes are arranged on either 96-well or 384-well plates to simultaneously characterize all 4 restriction digests from either one or 4 different DNA samples, respectively. The complete panels of 94 genes are arranged on 96- or 384-well plates to characterize one DNA sample either on 4 plates or all on the same plate, respectively. In addition to the 22 or 94 genes, each array contains 2 controls to monitor enzymatic digestion efficiency.
First, add equal amounts of each genomic DNA sample to components of the EpiTect Methyl II DNA Restriction Kit to set up 4 different restriction digests: mock (Mo), methyl-sensitive (Ms), methyl-dependent (Md), and double (Msd). After digestion and heat inactivation of the enzymes, mix each digest with the appropriate RT2 SYBR Green qPCR Mastermix, aliquot into the appropriate wells of the EpiTect Methyl II PCR Array, and run the recommended cycling program in your real-time PCR instrument.
Determine the CT values for the characterization of each digest with each gene-specific assay using your instrument’s software. Then, paste the values into the Excel-based data analysis template for the array format, in order to calculate the percentages of methylated and unmethylated DNA.
The EpiTect Methyl II PCR system is an innovative technology and versatile tool for:
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