SFRP1 methylation analyses of primary breast cancer specimens. Methylation-specific PCR (MSP) was performed on bisulphite-treated DNA from breast cancer (T) and matching normal primary breast tissue (N). MSP results from seven representative patients are shown. DNA bands in lanes labelled with U indicate PCR products amplified with primers recognising the unmethylated promoter sequence. DNA bands in lanes labelled with M represent amplified products with methylation-specific primers. Human placenta (PL) and in vitro methylated DNA (IVD) served as positive controls for the U and M reaction, respectively. Water was used as template in the negative control (NTC). Note that tumour tissue usually displayed a PCR product in the U-reaction as well, due to contaminating normal tissue (stromal cells, endothelial cells) present in the tumour specimens as has also been described by Suzuki et al. (2004).