Schematic of the procedure. Shown at the top is genomic DNA with a CpG island that is methylated. The DNA is cleaved with the restriction endonuclease MspI and adaptors ligated. The ligated material is divided evenly, one half being digested with McrBC and the other half being mock digested. This material is used as template for PCR amplification and the resulting product is used for microarray comparison.

The mean correlation for each sample with the normal samples (mean of 12 correlations for each tumor and 11 correlations for each normal) is shown. The 12 samples to the left of the vertical line are the normals. The 40 to the right are tumors. The horizontal line is drawn at the minimum mean correlation for the normals with normals. It is apparent that the tumors are on the whole distinct from the normals in comparison with each other (P < 10E–017).

Comparison of methylation analysis of cell lines. (A–C) shows the comparison of a normal fibroblast used to produce representations from two separate aliquots of DNA and the representations were analyzed and the intensity compared to each other by scatter plot; the intensities of each being on one and the other axis, and the correlation calculated: (A) being replicate hybs, (B) being technical replicates and (C) being biological replicates. (D) The comparison of the breast cancer cell line SKBR3 and the normal female fibroblast cell line as a scatter plot of the intensities from the SKBR3 experiment as the y-axis and the intensities of chp-skn-1 on the x-axis, and the correlation shown. (E) An example of bisulfite validation for a fragment found methylated in the tumor and not in the normal at a CpG island at chr 9:2197965–21980065, which lies upstream of the p14 gene. The original data showing the ratio differences is shown with the position of the fragment. To the right is the electropherogram identifying a region of cytosines that do not convert and at the bottom is a short table identifying the genomic position and number of McrBC site. (F) shows data for the cell line Huh7 compared to the normal cell line chp-skn-1 for the p16 gene CpG island graphed by genomic position. The x-axis is the genomic position of the probes and the y-axis is the ratio of two experiments, blue being chp-skn-1 and orange being Huh7. Inset above is shown is the relation of the transcript and inset below is the relation of the CpG island to the probes.

McrBC PCR of two different fragments of the MTSS1 CpG island for tumors identified of having methylation of this island compared to matched normal samples. Fragment 1 encompasses the MspI fragment we have identified as being methylated and overlaps the gene TSS. Fragment 5, we do not detect methylation. Both Normal and Tumor were digested with McrBC or mock digested for both matched pairs.