A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus

Abstract

Altered methylation patterns have been found to play a role in developmental disorders, cancer and aging. Increasingly, changes in DNA methylation are used as molecular markers of disease. Therefore, there is a need for reliable and easy to use techniques to detect and measure DNA methylation in research and routine diagnostics. We have established a novel quantitative analysis of methylated alleles (QAMA) which is essentially a major improvement over a previous method based on real-time PCR (MethyLight). This method is based on real-time PCR on bisulfite-treated DNA. A significant advantage over conventional MethyLight is gained by the use of TaqMan probes based on minor groove binder (MGB) technology. Their improved sequence specificity facilitates relative quantification of methylated and unmethylated alleles that are simultaneously amplified in single tube. This improvement allows precise measurement of the ratio of methylated versus unmethylated alleles and cuts down potential sources of inter-assay variation. Therefore, fewer control assays are required. We have used this novel technical approach to identify hypermethylation of the CpG island located in the promoter region of the retinoblastoma (RB1) gene and found that QAMA facilitates reliable and fast measurement of the relative quantity of methylated alleles and improves handling of diagnostic methylation analysis. Moreover, the simplified reaction setup and robustness inherent to the single tube assay facilitates high-throughput methylation analysis. Because the high sequence specificity inherent to the MGB technology is widely used to discriminate single nucleotide polymorphisms, QAMA potentially can be used to discriminate the methylation status of single CpG dinucleotides.

Figure 1

(a) Schematic of the QAMA. Bisulfite-treated target sequence is amplified with the same primer set (RBfw, RBrev) irrespective of its methylation status. Two differently labeled internal MGB TaqMan® probes (RBmet, RBunmet) bind their respective target and are cleaved by the 5′ nuclease activity of Taq DNA polymerase. The amount of fluorescence dyes VIC and FAM released during PCR is directly proportional to the amount of PCR product generated from the methylated or unmethylated allele, respectively. (b) Methylation analysis of a premixed sample with a methylation ratio of 50%. The relative fluorescence of VIC and FAM (delta Rn) is plotted as a function of the cycle number.

Figure 2

Standard curve as obtained by plotting the ΔC_T values against the predefined methylation ratio of each sample. The standard deviation for each sample analyzed in duplicate is indicated by error bars. The C_T value was set to 40 in the case that the respective fluorescence signal did not cross the threshold.

Figure 3

Test for inter-assay variability. Three independent assays, each represented by full, open or hatched bars were performed at different days. The methylation ratio of each sample was deduced from a standard curve running along with each assay.