Hello, we want to do methylation study with the Hıgh Resolution Melting Curve (HRM) method. Is there anyone using this method? Is there a study protocol you use? Thank you for your help and support.
Marios Didachos
Sample Preparation:Isolate genomic DNA from your sample of interest (e.g., tissue, blood, cells). Treat the DNA samples with bisulfite to convert unmethylated cytosines to uracil, while methylated cytosines remain unchanged. Primer Design:Design primers targeting the region of interest, which may include CpG islands or specific gene promoters known to undergo methylation changes. Ensure that the primers are specific to bisulfite-converted DNA sequences and avoid regions with repetitive sequences or SNPs. PCR Amplification:Perform PCR amplification using bisulfite-converted DNA as the template and methylation-specific primers. Optimize PCR conditions, including primer concentrations, annealing temperatures, and cycling parameters, to ensure specific and efficient amplification of the target region. HRM Analysis:After PCR amplification, subject the samples to high-resolution melting curve analysis using a real-time PCR instrument equipped with HRM capabilities. Gradually increase the temperature to denature the PCR products, while continuously monitoring fluorescence. Methylation-specific differences in DNA melting behavior will result in distinct melting curves, allowing for the detection of methylation patterns. Data Analysis:Analyze the HRM data to identify differences in melting curves between methylated and unmethylated samples. Use software or algorithms to normalize and compare the melting curves, and determine the methylation status of the target region based on curve shape, melting temperature (Tm), and other parameters. Validate the results using appropriate controls and reference standards, such as fully methylated and unmethylated DNA samples. Validation and Interpretation:Validate the methylation status of the target region using independent methods, such as bisulfite sequencing or methylation-specific PCR (MSP). Interpret the results in the context of your research objectives and biological significance, considering factors such as the location of methylation sites, their association with gene expression, and potential implications for disease or phenotype. Reporting and Documentation:Document the experimental procedures, including sample preparation, primer sequences, PCR conditions, and HRM analysis parameters. Present the results clearly and accurately, including graphical representations of melting curves and statistical analysis of methylation differences.
Performing a methylation study using the High-Resolution Melting Curve (HRM) method is a common and effective approach. HRM analysis is a sensitive technique that allows for the detection of DNA sequence variations, including methylation patterns, by monitoring changes in the melting behavior of double-stranded DNA fragments. Here's a general outline of how you can conduct a methylation study using HRM:
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