|Mixtures of methylated and unmethylated DNA (EpiTect Control DNA) of varying ratios were used as template. The average C T value was 24.94 with a standard deviation of only 0.05, equivalent to a CV of 0.2%. Human genomic DNA was used as template in 72 replicate real-time PCRs using a self-designed TaqMan® assay for BCL2 on the Rotor-Gene Q without ROX normalization. The average difference in the C T values between all dilutions was 1.07 cycles. Five replicate reactions were run for each dilution using a self-designed TaqMan® assay for IL1R2 and the Rotor-Gene Probe PCR kit on the Rotor-Gene Q. All pseudo-unknowns were correctly clustered according to genotype.|Twofold dilutions of human genomic DNA from 30 ng (10,000 copies) to 0.06 ng (20 copies) were used as a template in real-time PCR. A/T polymorphisms (class IV SNPs) are most difficult to discriminate due to minute differences between homozygote alleles (in this example, less than 0.1☌).
Data analysis was performed with the unsupervised mode of Rotor-Gene ScreenClust HRM Software. Experiments were performed using the Type-it HRM PCR Kit and a Rotor-Gene Q cycler with an HRM channel.