PAGE 7 OF 13 Nucleic Acids Research, 2008, Vol. 36, No. 7 e42 Figure 4. The quantitative accuracy of the SMART-MSP assays. The quantitative accuracy of the SMART-MSP technology was assessed using the 2( CT) quantiﬁcation approach. For each assay the calculated gene/control ratio for each standard is plotted against the dilution factor in a double logarithmic diagram. All assays proved to be quantitatively precise. (A) The CDH1 assay. (B) The DAPK1 assay. (C) The CDKN2A assay. (D) The RARB assay. higher GC-content, it is more stable and melted later than the bisulphite-modiﬁed amplicon. Identification of false positives in the CDH1 SMART-MSP assay By running the CDH1 SMART-MSP assay for an additional 10 cycles, late ampliﬁcation from the fully unmethylated control (WGA product) occurred. Since this WGA control is not methylated at the two CpG sites in between the primers, we expected to see a readily distinguishable left-shifted melting peak, and thus to be able to identify it as a false-positive result (Figure 1). The melting peak of the unmethylated control (WGA product) was shifted 1.28C to the left compared to the standards containing methylated template (Figure 7). Screening of cell lines The reliability of our method was tested using a panel of 14 cell lines (2008, MCF7, HS578T, MCF10A, MDA- MB-468, MDA-MB-231, MDA-MB-435, PC3, SKBr-3, Colo205, RPMI8226, SW948, HL-60 and T47D) with the CDH1 and CDKN2A SMART-MSP assays. Five of these cell lines were shown to be methylated at the CDH1 promoter at various levels (HL-60: 100%, MDA-MB-435: 75%, PC3: 10%, HS578T: 6% and RPMI8226: 2%). The ampliﬁcation data (Figure 8A) were used to calculate the methylation levels as described (see Materials and methods section). These results were validated using the CDH1 MethyLight assay, in which the same ﬁve cell lines were shown to be methylated at similar levels (Figure 8B). Our results were consistent with previously published data on these cell lines (28,29). However, the HS578T cell line was estimated to be methylated at lower levels when using the MethyLight assay relative to the SMART-MSP assay. This may be due to the fact that the probe in the MethyLight assay overlays two CpGs and that these are not consistently methylated in this cell line. The melting proﬁles obtained by the SMART-MSP assay was left-shifted, indicating that at least one of these two CpGs sites were unmethylated (data not shown). Five of the 14 cell lines were shown to be methylated by SMART-MSP at various levels at the CDKN2A promoter (T47D: 65%, PC3: 35%, RPMI8226: 30%, Colo205: 1% and SW948: 0.1%). Again, these results were consistent with previously published data on these cell lines (28,30). Screening of breast cancer samples The diagnostic applicability of the SMART-MSP meth- odology was tested using the RARB and CDH1 assays on a panel of 24 breast cancer samples. We found 6 out of the 24 samples to be methylated at the RARB promoter region at levels higher than 5% (76, 57, 44, 33, 29 and 7%). The ampliﬁcation data (Figure 8C) were used to calculate the methylation levels. Data from the control assay is not