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.

Article Wojdacz TK, Dobrovic A.. Methylation-sensitive high resoluti...

Article MethyLight: A high-throughput assay to measure DNA methylation

https://academic.oup.com/nar/article/33/21/6823/2401174

This link same your question

https://www.researchgate.net/post/How_to_estimate_DNA_methylation_using_real_time_PCR

Dear Vafadar

In the attached papers, real-time PCR assays were developed, that is well suited for this application and I hope to be useful to you.

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.

Article Wojdacz TK, Dobrovic A.. Methylation-sensitive high resoluti...

Article MethyLight: A high-throughput assay to measure DNA methylation

https://academic.oup.com/nar/article/33/21/6823/2401174

This link same your question

https://www.researchgate.net/post/How_to_estimate_DNA_methylation_using_real_time_PCR

Good luck.

HRM is a qualitative method and thus expressed as percentage

In the attached papers, real-time PCR assays were developed, that is well suited for this application and I hope to be useful to you.