Additional File 2.

CBFA2T3B promoter methylation levels examined using MSP The methylation levels in 24 breast tumor cell lines (labeled), 20 primary breast tumors (1T-20T), 20 normal breast counterparts (1N-20N) and 24 normal whole blood samples (1–24) are shown. Four separate regions (1–4) spanning 1-kb of CBFA2T3B promoter sequence were amplified using primers to detect for either unmethylated or methylated cytosines (see Figure 1B for primer locations and Additional file 7 for primer sequences and annealing temperatures). The asterisks indicate the samples examined by bisulfite sequencing. Unmethylated (U) and methylated (M) band intensities were scored as high (black dot), low (gray dot) or negative (white dot). Low-level basal methylation defined by high unmethylated to low methylated band intensities was detected in all normal bloods in ≥ 2/4 regions. High methylation was also detected in 21% of bloods in region four. Samples were considered hypermethylated when high intensity bands amplified in all four regions or hypomethylated when no methylation was detected. Based on this, 21% of breast tumor cell lines were hypermethylated (e.g. MDA-MB-231 and MDA-MB-468), 16% hypomethylated (e.g. BT-483 and MDA-MB-361) and 63% displayed a combination of basal to high methylation. Primary breast tumor samples were complex. 57% displayed basal methylation in ≥ 2/4 regions, 14% displayed high methylation in ≥ 2/4 regions and 29% tested negative in ≥ 3/4 regions. Normal breast counterpart samples were similar with 62% displaying basal methylation in ≥ 2/4 regions, 14% displaying high methylation in ≥ 2/4 regions and 24% testing negative in ≥ 3/4 regions. The detection of high-methylation in MCF-7 is due to spurious amplification events. Several overlapping primers spanning this region in this cell line were methylation negative (data not shown). This cell line is hypomethylated according to real-time MSP (pdf file).

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Bais et al. Molecular Cancer 2004 3:22   doi:10.1186/1476-4598-3-22