Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation

Abstract

In this article, we show that high resolution melting analysis (HRM) is a sensitive and specific method for the detection of methylation. Methylated DNA and unmethylated DNA acquire different sequences after bisulphite treatment resulting in PCR products with markedly different melting profiles. We used PCR to amplify both methylated and unmethylated sequences and assessed HRM for the determination of the methylation status of the MGMT promoter region. Reconstruction experiments showed that MGMT methylation could be detected at levels as low as 0.1%. Moreover, MS-HRM allows for estimation of the methylation level by comparing the melting profiles of unknown PCR products to the melting profiles of PCR products derived from standards with a known unmethylated to methylated template ratio. We used MS-HRM for the analysis of eight cell lines of known methylation status and a panel of colorectal cancer specimens. The simplicity and high reproducibility of the MS-HRM protocol makes MS-HRM the method of choice for methylation assessment in many diagnostic and research applications.

Figure 1.

The effect of annealing temperature on the sensitivity of the MS-HRM assay. The MGMT MS-HRM1 assay was run at the following annealing temperatures. (A) 60°C, (B) 62°C and (C) 63°C.

Figure 2.

The sensitivity of different MS-HRM assays for MGMT methylation. (A) MGMT MS-HRM1, (B) MGMT MS-HRM2 and (C) MGMT MS-HRM3. All the assays were run at the annealing temperature of 64°C which enables the highest sensitivity of methylation detection. The results from the 0.1% methylation dilution for MGMT MS-HRM1 were not reproducible between replicates and this dilution was excluded from the figure.

Figure 3.

Validation of the MGMT MS-HRM1 assay by the MGMT MethylLight assay. The samples are the series of dilution standards and three of the cell lines (MDA MB 468, SW480 and HS578T). Panel A shows the MGMT MS-HRM1 assay and panel B shows the MethylLight assay. The MS-HRM assay was run at an annealing temperature of 61°C.

Figure 4.

The MS-HRM assay for BNIP3 methylation. Results of the BNIP3-MS-HRM assay for five clinical samples compared to the dilution standards. Samples 1–5 show different methylation levels. The samples have been distributed over three panels to help distinguish the individual samples.