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.