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Cell Cycle EpiTect Methyl qPCR Array
The Human Cell Cycle EpiTect Methyl II Signature PCR Array profiles the promoter methylation status of a panel of 22 genes key to cell cycle regulation. Profiling cellular or fresh tissue genomic DNA samples with these arrays may help correlate CpG island methylation status with biological phenotypes. Inappropriate increases or decreases in the expression of cell cycle regulators by epigenetic mechanisms contribute to oncogenesis and senescence. Epigenetic regulation of cell cycle gene expression also occurs during development and the differentiation of a variety of cells from multi-potent progenitor cells to terminally differentiated non-dividing cells. This array contains genes that positively and negatively regulate the cell cycle, its phase transitions and checkpoints, DNA replication, and cell cycle arrest. The results may also help provide further insights into the molecular mechanisms and biological pathways behind cell cycle regulation. With a simple restriction enzyme digestion and real-time PCR, research studies can analyze the promoter methylation status of 22 different cell cycle regulators with this DNA methylation PCR array.
The EpiTect Methyl II PCR Arrays are intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Both 96-well and 384-well ( 4 X 96 ) formats are available
G1 Phase & G1/S Transition: CCND1, CCNE1, CDK4, CDKN1B (p27KIP1).
S Phase & DNA Replication: MCM2, MCM4.
G2 Phase & G2/M Transition: CCNB1, CDK5RAP1, CKS1B.
M Phase: CCNF, MRE11A, RAD51.
Cell Cycle Checkpoint & Cell Cycle Arrest: ATM, BRCA1, BRCA2, CDK2, CDKN1A (p21CIP1/WAF1), CDKN1B (p27KIP1), CHEK1, GADD45A, RAD9A, TP53.
Regulation of Cell Cycle: ATM, BRCA1, BRCA2, CCNB1, CCND1, CCNE1, CCNF, CDK2, CDK4, CDKN1A (p21CIP1/WAF1), CDKN1B (p27KIP1), CKS1B, GADD45A, RAD9A, RBL1, TP53
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.
What It Offers:
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.
For more detail on PCR Array layout, see the "Gene Table" link for the individual array products.
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.
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.
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
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.