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"If you have access to a real-time PCR instrument, you can analyze
the methylation status of multiple genes at the same time using the
EpiTect Methyl II PCR technology."
The EpiTect Methyl II PCR Array System is a fast, reliable technology that
profiles the DNA methylation status of a panel of genes. These PCR Arrays
can help you discover and verify cancer or developmental biomarkers useful
for both basic and applied research. No bisulfite conversion is required,
and the challenges of real-time PCR primers design and optimization has
already been done for you. EpiTect Methyl II PCR Arrays are ideal for fast,
high-throughput methylation analysis. For determining the exact methylation
level of individual and consecutive CpG sites, we recommend using bisulfite
Pyrosequencing on a PyroMark system.
You can use any 96-well or 384-well real-time PCR instrument.
Click below for detailed information.
How it Works
The EpiTect Methyl II PCR Array System 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. The high yield of DNA from the restriction digests and
PCR amplification allow the analysis of smaller, more heterogeneous samples.
Download User Manual
Tour the FREE Data Analysis
Template
What It Offers:
- Guaranteed Performance - Ready-to-use for DNA methylation analysis
- Time & Cost Savings - Less than 30 min hands-on time to
analyze up to 94 genes
- Easy Data Analysis - FREE Excel-based data analysis template
available
You can easily perform an EpiTect Methyl II experiment in your own
laboratory using any 96-well or 384-well real-time PCR instrument that you
have access to.
Or you can send your DNA samples to us and take advantage of our PCR Array
Services.
Array 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. The Signature panels of 22 genes are arranged on
either 96-well or 384-well plates to simultaneously characterize all four
restriction digests from either one or four different DNA samples,
respectively. The more Complete panels of 94 genes are arranged on 96- or
384-well plates to characterize all four restriction digests from one DNA
sample either on four plates or all on the same plate, respectively.
For more detail on PCR Array layout, see the "Gene Table" link
for the individual array products.
Data Interpretation
EpiTect Methyl II PCR Arrays provide gene methylation status as percentage
unmethylated (UM) and percentage methylated (M) fraction of input DNA.
"UM" represents the fraction of input genomic DNA containing no
methylated CpG sites in the amplified region of a gene. "M" represents
the fraction of input genomic DNA containing two or more methylated CpG sites in
the targeted region of a gene. The number of CpG sites methylated in a targeted
region can vary within the fraction of methylated input DNA.
In the figure above, each horizontal bar represents the targeted region of a gene from one genome. Biological samples usually contain many genomes derived from many cell types. For simplicity, five such genomes are depicted here. Light and dark circles represent unmethylated and methylated CpG sites, respectively.
Performance Data
To verify the reliability of the EpiTect Methyl II
PCR Array System, its results and sensitivity were compared with
bisulfite Sanger sequencing, the gold standard in DNA methylation
analysis Same Results as Bisulfite Sanger Sequencing
To validate the reliability of the system, EpiTect Methyl II PCR system
results (EpiTect Methyl II PCR) were compared with bisulfite Sanger
sequencing, the gold standard in DNA methylation analysis. The methylation
status of the cadherin 13 (CDH13) gene promoter was analyzed using either
bisulfite sequencing or EpiTect Methyl II PCR Assays in two different cell
lines. EpiTect Methyl II PCR Assays revealed that 100% of total input DNA had
methylated CDH13 gene promoter in MB231 cell lines whereas 100% of input DNA
from HeLa cell line had unmethylated CDH13 promoter. Importantly, EpiTect
Methyl II PCR Assays yielded results similar to Bisulfite Sequencing.
Same Sensitivity as Bisulfite Sanger Sequencing
Primary tumors are typically very heterogeneous, containing a mixture of
both cancerous and noncancerous cells. Therefore, reliable tumor
characterization requires detecting smaller amounts of methylated DNA diluted
in an unmethylated background. To test the sensitivity of EpiTect Methyl II
PCR system to detect methylated DNA diluted in an unmethylated background,
SKBR3 breast cancer cell line and normal blood genomic DNA (encoding
methylated and unmethylated HIC1, respectively) were mixed in different
ratios. EpiTect Methyl II Human HIC1 PCR Primers Assays were used to detect the
methylation status of the mixed sample. Result show that the percentage of
methylated HIC1 relative to total promoter DNA in each mixture was detectable
even down to six percent of the total DNA sample showing the high sensitivity
of EpiTect Methyl II PCR system.
Application Data
Gene promoter methylation is the most common epigenetic mechanism silencing
tumor suppressor genes during oncogenesis. Almost all cancer-related signaling
pathways are affected by methylation, and the number of genes affected in each
major type of cancer is still rapidly growing. However, even the most relevant
genes have not yet been correlated to individual cancer types or subtypes in
order to better define biological pathways and mechanisms leading to
oncogenesis and in order to properly develop DNA methylation biomarkers. The
EpiTect Methyl II PCR System provides an ideal reagent for such studies,
without bisulfite conversion of DNA. The following experiments demonstrate that
EpiTect Methyl II PCR Arrays can both verify known and discover new DNA
methylation cancer biomarkers
EpiTect Methyl II PCR Arrays are used to screen Breast Cancer Gene
Methylation Status in Breast Cancer Cell Lines.The heat map compares the
methylation status of 22 genes in the genomic DNA of three breast cancer cell
lines and blood genomic DNA (used as an unmethylated control) determined with
the Human Breast Cancer EpiTect Methyl II Signature PCR Arrays. The
results further strengthen the correlation of these biomarkers with breast
cancer.
Biomarker / Pathway Discovery
Cancer progresses through aberrant cell differentiation due to
alterations in gene expression. Transcription factors regulate gene
expression, and many tumor suppressor genes and oncogenes, defined by
classical genetic methods, encode transcription factors. Does the methylation
status of transcription factor genes differ between cancer and normal cells?
EpiTect Methyl II DNA Methylation PCR Arrays Discover New Candidate
Breast Cancer DNA Methylation Biomarkers. The heat map compares the
methylation status of a panel of 79 transcription factor genes in six
different breast cancer cell lines (some in duplicate) and a normal
epithelial cell line as determined using 384-well EpiTect Methyl II Custom
PCR Arrays. These breast cancer cell lines also methylate this gene panel
potentially providing a new source of cancer biomarkers. These results are consistent with the notions that aberrant expression of
transcription factors controlling cell differentiation plays key roles in
oncogenesis and that transcription factors can be tumor suppressors.
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