PAGE 3 OF 13 Nucleic Acids Research, 2008, Vol. 36, No. 7 e42 Table 1. Primer sequences, annealing temperatures, and amplicon information for the SMART-MSP assays (UCSC Genome Browser, November 2007) Gene CDH1 DAPK1 CDKN2A RARB COL2A1 Primer sequences (CpG sites in bold and converted Cs as capital Ts or As) F-gtgggcgggTcgtTagTtTc R-cgctAattAActAaAAAttcacctAccg F-aggaTagTcggaTcgagTTaacgTc R-ttAccgaAtcccctccgcgA F-gTaTTtTTtTcgagTaTtcgTtTacggc R-caaatcctctAAaAAAaccgcgA F-TcgagaacgcgagcgatTc R-gAccaatccaAccgAAAcg F-gTaatgTTaggagTaTTTtgtgggTa R-ctaccccaAAaAaAcccaAtcctA Annealing temperature (8C) 64 67 63 63 65 86 61 72 146 86 Amplicon size (bp) CpGs/non-CpG-Cs between the primers Spanned region 2/8 0/4 0/6 5/18 0/1 67328555-67328640 of Chr. 16 89302618-89302678 of Chr. 9 21964971-21965042 of Chr. 9 25444864-25445008 of Chr. 3 46667210-46667295 of Chr. 12 SMART-MSP primer design The primers were designed to include at least two CpG sites, and one of the cytosines of a CpG site was always placed at or adjacent to the 30 end. This will make the primers as selective for methylated templates as possible, and ensure that only these are ampliﬁed during PCR if suﬃciently stringent conditions are chosen. Non-CpG cytosines were included in the primer sequences as well to select against incompletely converted sequences, and at least one of these was placed as close to the 30 end as possible. Primer Express 1.5 (Applied Biosystems, Foster City, CA, USA) was used to calculate the estimated annealing temperature of the primers. Amplicons were designed to have only one melting domain according to the POLAND software (http://www.biophys.uni- duesseldorf.de/local/POLAND/poland.html) (25). The program Amplify (http://engels.genetics.wisc.edu/amplify/) was used to check for primer dimers, and no single nucleotide polymorphisms (SNPs) were found in any of the designed amplicons using BLAST searching of the SNP database (dbSNP BUILD 127). This is important since SNPs will interfere with the melting proﬁle (if found in between primers) or possibly aﬀect primer binding. The primer sequences and genomic regions spanned, as well as amplicon size and the annealing temperatures (TA) can be found in Table 1. PCR and HRM conditions for the SMART-MSP assays PCR cycling and HRM analysis were performed on the Rotor-Gene 6000TM (Corbett Research, Mortlake, Australia). SYTO 9 was used as the intercalating dye (Invitrogen, Carlsbad, CA, USA). The reaction mixtures consisted of 25 ng of bisulphite-modiﬁed template (the- oretical amount), 1x PCR buﬀer, 2.5 mmol/l MgCl2 ﬁnal (3 mmol/l in the CDH1 assay), 200 nmol/l of each primer, 200 mmol/l of each dNTP, 5 mmol/l of SYTO 9, 0.5 U of HotStarTaq (Qiagen) (5 U/ml) in a ﬁnal volume of 20 ml. The cycling protocol started with one cycle of 958C for 15 min for enzyme activation, followed by 45 cycles of 958C for 20 s, annealing at the appropriate temperature (Table 1) for 30 s, 728C for 30 s and one cycle of 958C for 1 min. HRM was performed from 608C to 908C, with a temperature increase at the rate of 0.28C/s for all assays. The annealing temperature was experimentally determined for each assay to ensure that only methylated templates were ampliﬁed. For each assay, a standard dilution series was run to assess the quantitative properties and sensitivity of these. Fully methylated and fully unmethyl- ated control (WGA product), unmodiﬁed control and no template control were also included in every run. All samples were analysed in triplicate and each breast tumour sample in duplicate. Gel electrophoresis analysis Five microlitres of the PCR products were resolved on 2.5% agarose gels and stained with ethidium bromide. The CDH1 and RARB MethyLight assays The same primers were used for the MethyLight assays as for the SMART-MSP assays. The probe sequences were FAM-50-ttcgcgttgttgattgg-30-BHQ for CDH1 and FAM- 50-ttgggtatcgtcggggta-30-BHQ for RARB. The reaction mixtures consisted of 25 ng of bisulphite-modiﬁed DNA (theoretical amount), 1x PCR buﬀer, 250 nmol/l of probe, 3 mmol/l MgCl2, 200 nmol/l of each primer, 200 mmol/l of each dNTP, and 0.5 U of HotStarTaq (Qiagen) (5 U/ml) in a total volume of 20 ml. The cycling protocol started with one cycle of 958C for 15 min, followed by 45 cycles of 958C for 20 s and 638C (648C in the CDH1 assay) for 40 s. PCR cycling was performed on the Rotor-Gene 6000. All samples were analysed in triplicate and each breast tumour sample in duplicate. Real time PCR quantification The relative 2( CT) quantiﬁcation approach (26) was used. The cycle threshold (CT) value of the control COL2A1 assay (Table 1) is subtracted from the CT value of the target gene for the calibrator sample, the 100% methylated standard. For each test sample, this value is then subtracted from the value resulting from the CT value of the target gene minus the CT value for the COL2A1 control assay. This gives the ‘ CT’ value, which is then put in to the equation, 2( CT), and multiplied by 100 to give the percentage of methylation relative to the 100% methylated control. For this approach to be valid, the ampliﬁcation eﬃciencies of the gene and the control must