Quantitative high-resolution melting analysis (HRM) with the Roche Lightcycler Instrument has been used for promoter methylation in analysis of formalin-fixed paraffin-embedded (FFPE) tissues.
Promoter hypermethylation is a frequent mechanism for the repression of gene transcription in cancer and is regarded as one of the hallmarks of cancer.
Analysis of DNA methylation is a promising tool for early cancer detection, risk assessment and response to therapy.
Since FFPE tissue samples are the largest source of material from normal controls and diseased tissues, their use is of high value for research.
Methodical evaluations are of high importance for demonstrating robustness and sensitivity of the assay, thus facilitating its establishment as a research tool and possibly a future routine test.
The aim of this study was to establish and evaluate HRM assays for detection of promoter methylation on archival FFPE tissues from individuals with colorectal cancer.
As proof of the principle, the researchers demonstrated the applicability of HRM for detection of promoter methylation using assays for O6-methylguanine-DNA methyltransferase (MGMT), adenomatous polyposis coli (APC), glutathione S-transferase P1 (GSTP1), and phosphatase and tension homolog deleted on chromosome 10 (PTEN) promoters in methylated DNA dilution matrix.
In a second step, HRM assays for MGMT and APC were tested on DNA isolated from fresh and FFPE human cancer cell lines.
These established MGMT and APC HRM assays were analysed using archival FFPE colorectal tumour specimens.
Methylated DNA levels as low as one per cent were reproducibly detected in a background of unmethylated DNA.
For certain applications, such as detection of rare events or risk stratification of individuals based on methylation status of specific markers, high sensitivity of the assay is important.
This sensitive assay can be adapted and used to detect low amounts of methylated cells within a tumour, or even to detect low numbers of tumour cells in the background of non-tumour cells in lymph nodes and other organs.
The most popular approaches for DNA methylation detection rely on the treatment of genomic DNA with sodium bisulfite, which converts cytosine into uracil while leaving 5-methyl cytosine unmodified.
This modification results in a sequence difference allowing for identification of methyl cytosines in a subsequent PCR amplification.
The most precise methylation profiling can be achieved by bisulfite sequencing, which allows identification of single methyl cytosines.
Several simpler PCR-based methods, which are especially important for small-scale research labs, have also been developed.
One of these newer and easier methods is high-resolution melting analysis (HRM), based on the 'melting' properties of DNA in solution.
The principle of this method is that bisulfite-treated DNA templates with different contents of methyl cytosine can be distinguished by melting analysis based on differences in melting temperatures.
HRM is a relatively simple and cost-effective method as it does not require expensive probes and reference gene assays for normalisation.
With HRM, all CpGs within the amplicon are analysed, enabling the assay to distinguish heterogeneous from homogeneous methylation by the shape of the melting curve.
This ability can be of importance, because methylation patterns at promoter CpG islands are typically not homogeneous.
