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EpiTect Methyl II PCR Assays

For DNA methylation profiling using MethylScreen technology with laboratory-verified assays

  • Experimentally verified real-time PCR primers
  • No bisulfite conversion required
  • Available for human, mouse, and rat samples
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.
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  • Entrez Gene IDs (e.g., 835)
  • RefSeq IDs (e.g., NM_032983, NP_116765)
  • Gene symbols (e.g., CASP2)
  • Cat. no. (e.g., SI00299551, QT01342509)
  • Sanger ID or Accession (e.g., hsa-let-7b, MI0000063)
  • CpG loci identification numbers (CG#) (e.g., CG17753661)

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EpiTect Methyl II PCR Assay
Laboratory-verified forward and reverse primers for 200 reactions
335002 Varies
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EpiTect Methyl II PCR Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

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).

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.

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, 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.

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.

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.


The EpiTect Methyl II PCR system is an innovative technology and versatile tool for:

Methylation pattern profiling
Verification of genomewide methylation analyses
Cancer and stem cell biomarker discovery
Toxicological and epidemiological screening
Characterization of gene expression regulation
Epigenetic DNA methylation analysis

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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