EpiTect Methyl II PCR Assays
For DNA methylation profiling using MethylScreen technology with laboratory-verified assays
EpiTect Methyl II PCR Assays are a simple and reliable method for quickly detecting the DNA methylation status of the CpG islands associated with individual genes, when used with the EpiTect Methyl II DNA Restriction Kit. EpiTect Methyl II PCR Assays are primer pairs available for analyzing any human, mouse, or rat promoter CpG island. EpiTect Methyl II PCR Assays use MethylScreen technology provided under license from Orion Genomics, LLC.
Performance
EpiTect Methyl II PCR Assays provide high sensitivity (see figure EpiTect Methyl II PCR Assays detect methylation in heterogeneous samples). Results are comparable to methylation analysis using bisulfite sequencing (see figure Comparison with bisulfite Sanger sequencing) and are highly suited for verification of genome-wide methylation analysis studies (see figure EpiTect Methyl II PCR Arrays generate data comparable to that from BeadChip platforms).
Principle
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 dinucleotides 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. EpiTect Methyl II PCR Assays are primer pairs available for more than 37,000 promoter CpG islands, annotated based on the combination of UCSC genome bioinformatics predictions and CpG islands with published evidence of hypermethylation. Each EpiTect Methyl II PCR Assay corresponds to one distinct CpG island in a promoter region, defined as from 5 kb upstream to 3 kb downstream of a transcription start site (TSS). Assays can be selected by gene, NCBI gene ID, or miRNA ID and are available for human, mouse, and rat. All EpiTect Methyl II PCR Assays are first designed by an experimentally optimized computer algorithm that accounts for the GC-rich sequences in genomic DNA and particularly in CpG islands. The design algorithm also ensures that every amplicon contains sufficient cutting sites for both methyl-sensitive and methyl-dependent enzymes to maximize methylation detection sensitivity. Assay designs are then tested experimentally for high amplification efficiencies and for amplification of a single, target-specific product to quality control real-time PCR performance. The performance of the EpiTect Methyl II PCR Assays is guaranteed when used with the appropriate RT2 SYBR® Green qPCR Mastermix.
Procedure
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) (see figure EpiTect Methyl II PCR Array procedure). After digestion and heat inactivation of the enzymes, add an aliquot of each digest to separate real-time PCR tubes containing the appropriate RT2 SYBR Green Mastermix and the EpiTect Methyl II PCR Assay. Finally, run the recommended cycling program.
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 correct Excel-based data analysis template for the array format to calculate the percentage of methylated DNA.
Applications
The EpiTect Methyl II PCR system is an innovative technology and versatile tool for:
Furthermore, EpiTect Methyl II PCR Assays are also powerful tools for studying regulatory mechanisms behind the gene expression changes observed with RT2 Profiler PCR Arrays and Assays.
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