Mixtures of methylated and unmethylated DNA (EpiTect Control DNA) of varying ratios were used as template. A CpG island from the promoter region of the APC gene (adenomatosis polyposis coli) was amplified and the degree of methylation was determined by HRM methylation analysis on the Rotor-Gene Q using the EpiTect HRM PCR Kit. [A] A standard normalized melt curve and a [B] difference plot normalized to the 50% methylated sample are shown.
A human A/T SNP in the AHRR7 gene was analyzed using genomic DNA from wild-type (blue), homozygous mutant (green), and heterozygous (red) samples. Experiments were performed using the Type-it HRM PCR Kit and a Rotor-Gene Q cycler with an HRM channel. Data analysis was performed with the unsupervised mode of Rotor-Gene ScreenClust HRM Software. 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°C). [A] HRM raw data, [B] cluster plot. All pseudo-unknowns were correctly clustered according to genotype.
For use with the Rotor-Gene Q for outstanding performance in real-time PCR
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. 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. The average difference in the CT values between all dilutions was 1.07 cycles. 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 CT value was 24.94 with a standard deviation of only 0.05, equivalent to a CV of 0.2%.