EpiTect Methyl II PCR Arrays

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

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. 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.

Array formats

EpiTect Methyl II PCR Array Format A: 96-well plates containing dried qPCR primer assays, Optical Thin-Wall 8-Cap Strips

EpiTect Methyl II PCR Array Format C: 96-well plates containing dried qPCR primer assays, Optical Adhesive Film

EpiTect Methyl II PCR Array Format D: 96-well plates containing dried qPCR primer assays, Optical Thin-Wall 8-Cap Strips

EpiTect Methyl II PCR Array Format E (Signature Panels): 384-well plate(s) containing dried qPCR primer assays, Optical Adhesive Film, 384EZLoad Covers

EpiTect Methyl II PCR Array Format E (Complete Panels): 384-well plate(s) containing dried qPCR primer assays, Optical Adhesive Film

EpiTect Methyl II PCR Array Format F: 96-well plates containing dried qPCR primer assays, Optical Adhesive Film

EpiTect Methyl II PCR Array Format G (Signature Panels): 384-well plate(s) containing dried qPCR primer assays, Optical Adhesive Film, 384EZLoad Covers

EpiTect Methyl II PCR Array Format G (Complete Panels): 384-well plate(s) containing dried qPCR primer assays, Optical Adhesive Film, Optical Adhesive Film

Cancer-focused arrays

Breast cancer

Cancer miRNA

Colon cancer

Epithelial-to-mesenchymal transition (EMT)

Gastric cancer

Leukemia and lymphoma

Liver cancer

Lung cancer

Melanoma

Prostate cancer

Tumor suppressor genes

Pathway-focused arrays

Apoptosis

Cell cycle

Cytokine production

DNA repair

Homeobox (HOX) genes

Inflammatory response and autoimmunity

Mental disorders

Notch signaling pathway

Polycomb & trithorax complexes

Stem cell transcription factors

Stress & toxicity

T cell and B cell activation

T helper cell differentiation

TGF-β/BMP signaling

Toll-like receptor signaling

Tumor suppressor genes

WNT signaling

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 C_T 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:

Methylation pattern profiling

Validation of genomewide methylation analyses

Cancer and stem cell biomarker discovery

Toxicological and epidemiological screening

Characterization of gene expression regulation

Epigenetic DNA methylation analysis