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PDF file - 348KB, The semiquantitative methylation analysis and pyrosequencing in the methylation-variable transitional-CpG sites. (A) Schematic sequence lines of transitional-CpG sites near the PPARG, CDKN2A, CDH1, and TFF2 genes. PCR primers for the MSP are indicated by the boxes above the vertical lines marking the individual CpG sites. (B) The isotope-labeling semiquantitative MSP of representative samples. The lane marked by U and M indicate the PCR bands of the unmethylation and methylation primer sets, respectively. The methylation level of each sample is indicted below the lanes. (C) Pyrosequencing for the methylation-variable sites. Quantitative pyrosequencing of the PPARG and CDKN2A genes was not possible because common PCR generated non-specific bands over a wide range of annealing temperatures. In cases of CDH1 and TFF2 genes, agarose gel electrophoresis of the pyrosequencing-PCR products showed a main band corresponding to expected size of PCR amplicons. Pyrosequencing could be performed using the same genomic DNAs analyzed by semiquantitative MSP. The relative density of methylation (percent) analyzed by pyrosequencing is illustrated as plots. The methylation values of the semiquantitative MSP and pyrosequencing methods were similarly ranked in ascending order as follows: level 1 vs. 9percent; level 2 vs. 11percent; level 3 vs. 19percent; level 5 vs. 32percent and 36percent; level 6 vs. 45percent; level 7 vs. 56percent and 63percent. The shaded areas correspond to CpG loci in which the intensity ratio of nucleotide C-to-T is measured as relative densities (percent) of methylation of the loci. The arrows indicate bands at the expected size of PCR amplicons for PPARG (167 bp), CDKN2A (141 bp), CDH1 (135 bp), and TFF2 (132 bp) genes. M, 100 bp molecular weight marker. |
