e42 Nucleic Acids Research, 2008, Vol. 36, No. 7 PAGE 6 OF 13 Figure 3. The sensitivity of the SMART-MSP assays. In all assays, the 0.1% methylated standard could be detected with high reproducibility. (A) The CDH1 assay. (B) The DAPK1 assay. (C) The CDKN2A assay. (D) The RARB assay. Sensitivity and quantitative accuracy of the SMART-MSP assays Each SMART-MSP assay was optimized so that ampli- ﬁcation only occurred from standards containing methyl- ated template, and no ampliﬁcation was seen in the fully unmethylated control (WGA product), in the unmodiﬁed control or in the no template control. The sensitivity and quantitative accuracy of the assays were determined using the standard dilution series (see Materials and methods section). All our assays were able to reliably detect the 0.1% methylated standards (Figure 3). The quantitative accuracy of each assay was determined using the relative 2( CT) quantiﬁcation approach (see Materials and methods section). Ampliﬁcation eﬃciencies of the gene and the control were approximately equal for all assays (data not shown). The calculated values for each standard were plotted versus the dilution factor for each gene (Figure 4). All reactions contained approximately equal amounts of template suitable for PCR. This was evident from the similar CT values obtained in the control assays (data not shown). We used the software to obtain a standard curve for the dilution series for calculation of the correlation coeﬃcient (r2) for each assay. The correlation coeﬃcient for each assay (DAPK1: r2 = 0.995, CDKN2A: r2 = 0.998, CDH1: r2 = 0.999, RARB: r2 = 0.995) indicated a strong linear relationship between CT values and given concen- trations for all assays. We also tested the CDH1 and RARB MethyLight assays. These MethyLight assays were quantitatively accurate in the range from 100% down to 0.1% methyl- ated template. The correlation coeﬃcients of the MethyLight assays were: CDH1: r2 = 0.984 and RARB: r2 = 0.983, again indicating a strong linear relationship between CT values and given concentrations for both. Validation of the DAPK1 and CDKN2A SMART-MSP conversion control assays Bisulphite conversion can be assessed by HRM analysis using assays with non-CpG cytosines between the primers. If a right shift of the melting proﬁle is observed, this can only be due to incomplete conversion of some or all of the non-CpG cytosines in between the primers or ampliﬁca- tion of non-speciﬁc products (Figure 1). Since incom- pletely converted products are of the same size as true positives, these cannot be distinguished using gel electro- phoresis. We generated incompletely converted DNA (see Materials and methods section) to assess whether its ampliﬁcation showed right-shifted melting proﬁles in these assays. Ampliﬁcation was usually seen from these samples and always showed right-shifted melting proﬁles. As expected, the 100% methylated standard ampliﬁed earlier than incompletely converted DNA in both assays, and thus gave higher melting peaks (Figure 5). We showed that bisulphite-modiﬁed template melts early relative to unmodiﬁed template (Figure 6). A primer pair (50-gggaagatgggatagaagggaataT and 50-tctAacaAttA tAAActccaaccaccaa) with a limited number of non-CpG cytosines in their sequences (shown in upper case), and thus not particularly discriminatory against unmodiﬁed sequences, was used to amplify bisulphite-modiﬁed as well as unmodiﬁed DNA from the same sample. In this assay, ﬁve non-CpG cytosines and no CpG sites are found in between the primers. Since the unmodiﬁed amplicon has a