EpiTect Methyl II PCR Assay Service
For profiling methylation status of individual genes using MethylScreen technology
QIAGEN’s EpiTect Methyl II PCR Assay Service offers DNA methylation profiling services using the EpiTect Methyl II PCR system. This innovative technology enables fast and accurate CpG island DNA methylation profiling of individual genes. EpiTect Methyl II PCR Assays use MethylScreen technology provided under license from Orion Genomics, LLC.
EpiTect Methyl II PCR Assays rely 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 the relative amount of DNA remaining after each enzyme digestion is measured by real-time PCR, the methylation status of individual genes and the methylation profile across a gene panel are reliably and easily calculated. The high yield of DNA from the restriction digests and PCR amplification allow the analysis of smaller, more heterogeneous samples.
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 the transcription start site (TSS). Assays are available for about 13,000 promoter CpG islands, annotated according to the UCSC genome bioinformatics predictions in addition to published evidence. All EpiTect Methyl II PCR Assays are first designed by a rigorously optimized algorithm that accounts for the GC-rich sequences in genomic DNA and in CpG islands. The design algorithm also ensures that every amplicon contains sufficient cutting sites for both methyl-sensitive and methyl-dependent restriction enzymes to maximize methylation detection sensitivity. Verified and optimized processes yield the highest data quality and rapid turnaround time.
Four different restriction digests are carried out on a DNA sample: methylation-sensitive enzyme, methylation-dependent enzyme, double digest with both enzymes, and a mock digest with no enzyme. Next, quantitative real-time PCR of genes of interest detect the amount of DNA remaining in each reaction. The methylation status — methylated, hypermethylated, or unmethylated — is calculated from these amounts.
For instructions regarding sample submission and shipping, please contact QIAGEN Technical Services. Samples accepted are cells or tissue or total DNA. For cells and tissue, Service Core offers a DNA isolation service.
For all samples
All sample collection should be carried out in a timely manner in a DNase-free environment. Please note the minimum amount of starting materials required (see below for requirements for each sample type). If you have less than the minimum amount or your previously isolated samples have been stored differently than we recommend, please contact QIAGEN Technical Services before submitting your samples.
Provide at least 0.5 million cells for each sample (specify number of cells on the submission form). Snap freeze the cell pellets in liquid nitrogen, store samples frozen at –80°C, and ship on dry ice.
Provide at least 50 mg of tissue for each sample (specify the weight of the tissue on the submission form). Snap freeze tissues in liquid nitrogen. Store samples frozen at –80°C, and ship on dry ice.
Purified genomic DNA
Purified genomic DNA is required for methylation analysis. A genomic DNA isolation service is also available, please inquire.
Genomic DNA isolated using a standard genomic DNA isolation kit, such as the DNeasy Blood & Tissue Kit, which is compatible with methylation profiling. We recommend 2 µg of DNA per sample (in DNase-free water or TE buffer) at a concentration ≥30 ng/µl. A260/A280 ratio of ≥1.7 and A260/A230 ratio of ≥1.7 is required. Customers should check sample integrity by agarose gel electrophoresis before sending samples to QIAGEN. sample gel pictures, if available, should accompany the submitted samples.
Samples should also be accompanied by a sample submission sheet. Excel format is preferred for sample submission, and the spreadsheet should contain the following information: sample ID, concentration, A260/A280, A260/A230, total volume, total DNA amount.
QIAGEN will measure DNA concentration and assess the quality of the submitted DNA using an Agilent Bioanalyzer.
Other sample types
Please contact QIAGEN Technical Services.
DNA samples are processed by QIAGEN for EpiTect Methyl II PCR Assays using the following steps:
Data in an electronic form can be downloaded from a secure FTP website or delivered via email. A complete data and experiment report includes:
EpiTect Methyl II PCR Assays can reliably and sensitively profile DNA methylation status for the discovery and development of biomarkers important to stem cell growth and differentiation, cancer, and other human diseases.