e42 Nucleic Acids Research, 2008, Vol. 36, No. 7 PAGE 4 OF 13 Region in between primers Non-CpG cytosines CpG (and non-CpG) cytosines Comments Melting profile (theoretical) Melting profile (theoretical) Amplification Early * Intermediate to late Very late Late M M† M ( )‡ Amplification Early * Intermediate to late Very late Late * Fully methylated template will amplify early relative to a heterogeneously methylated template. However, late amplification is observed when the percentage of fully methylated template is low. † In this situation,a complex melting pattern composed of heteroduplexes as well as homoduplexes will generally occur. The more CpGs in between the primers, the more complex the pattern. ‡ A single left-shifted melting peak is an indication that all the CpGs in between the primers were unmethylated, and thus amplification may be a result of false priming. § If non-CpG cytosines are included a right-shifted melting profile may be observed, dependent on the methylation status of the CpG cytosines in between the primers. This can be complex to interpret. Full methylation Heterogeneous methylation Unmethylated (false priming) Incomplete conversion N N N N ( ) M ( )§ Figure 1. A schematic overview of SMART-MSP. Bisulphite-modiﬁed DNA is ampliﬁed in real time using a HRM-compatible intercalating dye to obtain quantitative data. After real-time PCR a HRM step is performed for quality control of the amplicon. The interpretation is made by considering both the real-time PCR and the HRM information. Two diﬀerent types of SMART-MSP amplicon design are shown here, in combination with the melting proﬁles and ampliﬁcation data that can be expected (vertical rows), in diﬀerent methylation and conversion situations (horizontal rows). Incomplete conversion can be detected most readily when non-CpG cytosines are found in between the primers and no CpG cytosines are found. By including CpG cytosines in between the primers, it can be determined if the region is heterogeneously methylated or unmethylated. If the CpG cytosines in between the primers are unmethylated, the ampliﬁcation might be a result of false priming. N and M are theoretical temperatures dependent on the amplicon size and sequence. be approximately equal (26). The take-oﬀ values given by comparative quantiﬁcation (a feature of the Rotor-Gene 6000 Series Software, version 1.7.61) were used as CT values in the calculations. The take-oﬀ point is deﬁned as the cycle at which the second derivative is at 20% of the maximum level, which indicates the end of the back- ground noise and the transition into the exponential phase. RESULTS The basis of SMART-MSP Real-time ampliﬁcation of bisulphite-converted DNA with MSP primers is performed with a ﬂuorescent dye, which does not inhibit the PCR when used at saturat- ing conditions (22). This allows for highly accurate quantitative results to be obtained without the use of probes and for HRM analysis to be performed. Quantiﬁcation is based on CT values, and thus it is necessary to run a control assay in parallel to normalize for the amount of input DNA in the PCR. We designed a new COL2A1 control assay to estimate the amount of ampliﬁable template (Table 1), adjacent to the region used previously as an input control in MethyLight-based assays (27). The region we chose has improved selection against the ampliﬁcation of unconverted DNA (data not shown). In the SMART-MSP methodology, sensitive melting analysis using HRM is performed immediately after the real-time PCR in a closed-tube system. The kind of information that can be obtained from HRM is dependent on amplicon design, and should be interpreted by considering the ampliﬁcation data as well (Figure 1). First, when only non-CpG cytosines are included in