EpiTect Methyl II PCR Arrays allow the simultaneous
DNA methylation profiling of a panel of 22 or 94 genes promoters using
restriction enzyme based MethylScreen™ technology. Genes are carefully selected
based on their reported methylation statusin a variety of experimental
settings. These arrays allow correlation of CpG island methylation status with
biological phenotypes or disease outcomes. Both EpiTect Methyl II Signature PCR
Arrays (22 genes) and EpiTect Methyl II Complete PCR Arrays (94 genes) are
available for human, mouse or rat studies in 96- or 384-well formats.
With
a simple restriction enzyme digestion followed by real-time PCR, you can
simultaneously analyze the DNA methylation levels of 22 or 94 different disease-
or pathway-focused genes.
The method is based on the detection of remaining input DNA after
cleavage with a methylation-sensitive (MSRE) and/or a methylation-dependent (MDRE)
restriction enzyme. These enzymes will digest unmethylated and methylated
DNA, respectively. Following digestion, the remaining DNA is quantified by
real-time PCR in each individual enzyme reaction using primers that flank a
promoter (gene) region of interest. The relative fractions of methylated and
unmethylated DNA are subsequently determined by comparing the amount in each
digest with that of a mock (no enzymes added) digest using the ΔCt method. The
reliability and simplicity of the procedure make this technology highly
suited for semi-high-throughput DNA methylation profiling and biomarker
discovery for various research fields, such as stem cell differentiation
and development.
Input genomic DNA is aliquoted into four equal portions and subjected to mock
(Mo), methylation sensitive (Ms), methylation dependent (Md), and double (Msd)
restriction endonuclease digestion. After digestion, the enzyme reactions are
mixed directly with qPCR master mix and aliquoted into a PCR Array plate
containing pre-dispensed primer mixes. Run real time PCR using specified cycling
conditions. Finally, copy and paste raw Ct values to data analysis excel sheet
which automatically calculates the relative amount of methylated and
unmethylated DNA fractions. Learn More.
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
22
or 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
Similar Results to Bisulfite Sequencing
To validate the reliability of the system, EpiTect Methyl II PCR system results (EpiTect Methyl II PCR) were compared with bisulfite conversion, the gold standard in DNA methylation analysis (Bisulfite conversion). The methylation status of the cadherin 13 (CDH13) gene promoter was analyzed using either bisulfite conversion or EpiTect Methyl II PCR Assays in two different cancer 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
High Sensitivity
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 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
Screening for DNA methylation biomarkers:
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
cancer is growing rapidly. In order to better define biological pathways and
mechanisms leading to oncogenesis, the DNA methylation profiling of a large
number of genes is required. The EpiTect Methyl II PCR Array System provides an
ideal tool for analyzing DNA methylation of multiple genes in multiple samples,
simultaneously, without any bisulfite conversion of DNA. To demonstrate this
application, methylation status of 79 transcription factor genes were studied
in six breast cancer lines and a normal epithelial cell line with Custom
EpiTect Methyl II PCR Arrays.
The methylation status of a panel of 79 transcription factor genes in six
breast cancer lines and a normal epithelial cell line is shown as heat map.
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.
Moreover, it confirms the ability of EpiTect Methyl II PCR Arrays to discover
new DNA Methylation biomarkers.
Validation of genome-wide methylation analysis:
EpiTect Methyl II PCR system is best fit for validating the results of
genome-wide methylation analysis studies. To demonstrate this application,
the methylation pattern of 14000 genes was studied in MCF7 cells using
Illumina® Infinium® Assay (HumanMethylation27 BeadChip). Results from this
Chip were further validated with the EpiTect Methyl II Complete Human Breast
Cancer PCR Array
EpiTect Methyl II PCR Array (Human Breast Cancer PCR Array, Complete Panel) validated the methylation status of 94 genes with a single PCR run. The methylation pattern of genes was consistent with that observed with Illumina® Infinium® Assay. A representative comparison of 15 genes is shown. For better comparison, results from the EpiTect Methyl II PCR Array were converted to averaged Beta values, where 0 means completely unmethylated DNA while 1 means completely methylated DNA.
The EpiTect Methyl II PCR Arrays use the MethylScreen™ Technology provided under license from Orion Genomics, LLC.
Complete Array List
