EpiTect Methyl II Custom PCR Arrays

For profiling DNA methylation of user-defined gene panels using MethylScreen technology
  • Experimentally verified real-time PCR primers
  • Ready-to-use for DNA methylation analysis
  • Less than 30 minutes hands-on time
  • No bisulfite conversion required
  • Available for human, mouse, or rat samples
EpiTect Methyl II Custom PCR Arrays allow you to define your own gene list, tailored to your specific research interest. We then find the nearest predicted CpG islands or other differentially methylated regions closest to those selected target genes to identify the required cataloged real-time PCR assays. Rigorous PCR primer verification and quality controlled manufacturing guarantee reliable assay performance. Build your EpiTect Methyl II Custom PCR Arrays using our genomewide DNA methylation primer assays. EpiTect Methyl II Custom PCR Arrays use MethylScreen technology provided under license from Orion Genomics, LLC.
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EpiTect Methyl II Custom PCR Arrays
For methylation analysis of a custom panel of human, mouse, or rat genes in 96-well or 384-well plates
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EpiTect Methyl II Custom PCR Arrays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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EpiTect Methyl II PCR Assay detects methylation in heterogeneous samples.|Comparison with bisulfite Sanger sequencing.|Schematic view of the EpiTect Methyl II PCR Array System procedure.|Screening for DNA methylation biomarkers.|EpiTect Methyl II PCR Array formats.|EpiTect Methyl II PCR Arrays generate data comparable to that from BeadChip platforms.|Pictorial explanation of EpiTect Methyl II PCR Assay results.|
Analytical sensitivity was tested using a serial dilution of SKBR3 genomic DNA and peripheral blood leukocyte genomic DNA. Using Human HIC1 DNA EpiTect Methyl II PCR Primers, the percentage of methylated HIC1 relative to the total amount of input DNA was detectable even down to only 6% of the total DNA sample. The HIC1 gene promoter is methylated in cancer cells and unmethylated in normal cells.
|Methylation in the CDH13 promoter region was analyzed using EpiTect Methyl II PCR Assays or bisulfite Sanger sequencing. EpiTect Methyl II PCR Assays were performed using the EpiTect Methyl II PCR Assay Handbook protocol. For bisulfite sequencing, genomic DNA was bisulfite converted and amplified with gene-specific methylation-independent primers that included the amplicon region designed for the EpiTect Methyl II PCR Assay. PCR products were purified and sub-cloned; 16 colonies for each gene in each cell line were sequenced.|The system relies on the differential cleavage of target sequences by two different restriction endonucleases. qPCR allows the analysis of the methylation status of up to 94 targets simultaneously. SEC and DEC are control assays for monitoring enzymatic activities of restriction digestion enzymes.|The methylation status of 79 transcription factor genes in 6 breast cancer lines and a normal epithelial cell line is shown as heat map. These results are consistent with the idea that aberrant expression of transcription factors controlling cell differentiation plays key roles in oncogenesis and that transcription factors can be tumor suppressors, and confirms the ability of EpiTect Methyl II PCR Arrays to discover new DNA methylation biomarkers. |The flexible format of Custom EpiTect Methyl II PCR Arrays can accommodate a range of experimental designs, from testing the same site in many samples to testing one sample for 96 different methylation sites. |EpiTect Methyl II PCR Array and Illumina Infinium Human Methylation 27 BeadChip assays were performed on MCF-7 cells. A representative analysis of 22 genes is shown. For better comparison, results from the EpiTect Methyl II PCR Array were converted to averaged beta values, in which 0 means completely unmethylated and 1 means completely methylated.
|EpiTect Methyl II PCR Assays and Arrays provide gene methylation status as percentage unmethylated (UM) and percentage methylated (M) fraction of input DNA. In these examples, the horizontal bar represents the targeted region of a gene from one genome. Biological samples usually contain many genomes derived from many cell types; here, five such genomes are depicted. Light and dark circles represent unmethylated and methylated CpG sites, respectively. In example 2, the targeted region of a gene has two or more methylated CpG sites in two out of five genomes. Thus, the EpiTect Methyl II PCR Assay data reveal that this gene is 60% unmethylated and 40% methylated.
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Performance
EpiTect Methyl II Custom 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 qPCR Arrays are highly suited for biomarker discovery studies (see figure Candidate breast cancer DNA methylation biomarkers).
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.

The EpiTect Methyl II PCR Array system, using MethylScreen technology, relies on the differential cleavage of target sequences by 2 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.

QIAGEN's proprietary bioinformatics algorithm reliably selects which promoter CpG islands will most likely represent the DNA methylation status of your custom gene list. The rigorous wet-bench PCR primer verification and quality-controlled manufacturing ensure high levels of CpG island-specific assay specificity and amplification efficiency. The performance of the assays on the EpiTect Methyl II Custom PCR Arrays is guaranteed when used with the appropriate RT2 SYBR® Green qPCR Mastermix.

EpiTect Methyl II Custom PCR Arrays allow the simultaneous DNA methylation profiling of a panel of gene promoters. Arrays are available in both 96- and 384-well plate formats to analyze the methylation status of up to 24 or 96 genes related to a specific area of research. Simply select the genes and plate format required (see figure EpiTect Methyl II PCR Array formats) and then contact us.

Procedure

First, add equal amounts of each genomic DNA sample to components of the EpiTect Methyl II DNA Restriction Kit to set up four 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 on a 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 your array format to calculate the percentage of methylated DNA.

Applications
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 Arrays 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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Kit Handbooks
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(EN) - EpiTect Methyl II PCR Array Handbook
For pathway- or disease-focused profiling of regional DNA methylation using MethylScreen technology
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