High-Resolution Melting Analysis Using the Roche LightCycler Instrument
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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.
In a recent study, quantitative high-resolution melting analysis (HRM) with the Roche LightCycler Instrument was used for promoter methylation for analysis of formalin-fixed paraffin-embedded (FFPE) tissues.
Since FFPE tissue samples are the largest source of material from normal controls and diseased tissues, their use is of inestimable 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 the 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 analyzed using archival FFPE colorectal tumor specimens. Methylated DNA levels as low as 1% were reproducibly detected in a background of unmethylated DNA.
In a recent study, quantitative high-resolution melting analysis (HRM) with the Roche LightCycler Instrument was used for promoter methylation for analysis of formalin-fixed paraffin-embedded (FFPE) tissues.
Since FFPE tissue samples are the largest source of material from normal controls and diseased tissues, their use is of inestimable 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 the 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 analyzed using archival FFPE colorectal tumor specimens. Methylated DNA levels as low as 1% were reproducibly detected in a background of unmethylated DNA.
