PAGE 9 OF 13 Nucleic Acids Research, 2008, Vol. 36, No. 7 e42 Figure 7. Identiﬁcation of false positives in the CDH1 SMART-MSP assay. The CDH1 SMART-MSP assay was performed with an additional 10 cycles to obtain ampliﬁcation from the unmethylated control (WGA product) shown in green. (A) Real-time PCR ampliﬁcation data. (B) The melting peak of the fully unmethylated control was left-shifted by 1.28C relative to melting peaks of the standards containing methylated template, and could thus be identiﬁed as a false-positive result. Figure 8. Screening of cell lines for CDH1 methylation and breast cancer samples for RARB methylation. (A) CDH1 SMART-MSP ampliﬁcation data for the positive cell lines. Five out the 14 cell lines screened were shown to be methylated at the CDH1 promoter. (B) CDH1 MethyLight ampliﬁcation data from the positive cell lines. The data from the MethyLight assay was consistent with the data from the SMART-MSP assay. (C) RARB SMART-MSP ampliﬁcation data for the positive tumour samples. Six out the 24 samples screened were shown to be methylated at the RARB promoter. (D) RARB MethyLight ampliﬁcation data from the positive tumour samples. The data from the MethyLight assay was consistent with the data from the SMART-MSP assay. the most sensitive non-quantitative technique available and can detect 0.1% methylation or less. A major drawback of MSP is its susceptibility to false positives (12–14). This can be due to incomplete conver- sion of the template, false priming or a sensitivity issue due to the capacity of MSP to amplify very low-level methylation. Incomplete bisulphite conversion is probably the most problematic cause of false-positive results. When perform- ing MSP experiments, it is thus important to be conﬁdent that positive results are not derived from incomplete conversion. MSP primers are normally designed to have two or preferably more cytosines deriving from CpG sites at or near the 30-end. This makes the primers highly