Guidelines for successful HRM
Successful HRM requires five steps – from PCR to result
Achieve success in your HRM experiment by following these simple guidelines:
- Ensure that the amplification was successful, resulting in specific products and no artifacts such as primer dimers. A sigmoidal amplification plot and comparable CT values for all samples indicate successful amplification.
- Amplification specificity can be optionally confirmed by classical melt curve analysis. Presence of a single melt peak indicates the presence of a specific PCR product and absence of PCR artifacts or byproducts such as primer dimers. However, when examining mixtures of methylated and unmethylated DNA samples, or when an amplicon has a complex composition comprising of different domains that melt at different temperatures, a single, distinct peak is not observed.
- Before HRM curves are plotted, the data from samples (excluding no template control [NTC], nonspecific PCR products, etc.) is first normalized. This is because comparison of HRM curves can only occur if all samples are on the same scale. However, since HRM curves can have different starting points, meaning the scale of each melt curve can be different, normalization is required.
- A reference genotype is selected and serves as a reference for the difference plot, and all samples are compared to this genotype. The melt curve of each sample is overlaid and confidence intervals are generated relative to the known genotype.
- Based on the genotypes defined, unknown genotypes will either be categorized as related to known genotypes or will be marked as variations.
Analyze small DNA fragments
Analyze a single, pure product
Use sufficient pre-amplification template
Check for aberrant amplification plots
Keep the post-amplification sample concentrations similar
Ensure sample-to-sample uniformity
Allow sufficient data collection for pre- and post-melt phases
Use a standardized DNA purification method for all samples
It is recommended to use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids introduction of variations due to differing compositions of elution buffers used in different extraction methods.
- To avoid any reduction in performance, we recommend using QIAGEN genomic DNA purification kits such as QIAamp or DNeasy Kits.
- Use 1 ng to 50 ng of template genomic DNA or 1–50 pg microbial DNA per 25 μl reaction.
- Use comparable amounts of template genomic DNA for all samples resulting in CT values below 30 and differing by no more than three CT values.
- It is recommended to use control DNA and sample DNA of comparable integrity. For example, if analyzing samples from FFPE tissues, control DNA should also be derived from FFPE tissues with comparable integrity.
- DNA samples used for HRM should be normalized in concentration. All DNA samples should be quantified and then adjusted to the same concentration using the same dilution buffer. Use sufficient PCR cycles so that all samples have reached the plateau phase of PCR to ensure that comparable amounts of PCR product are generated. Note that the amount of DNA affects the melting temperature of the PCR product.
If performing methylation analysis, ensure that a standardized bisulfite conversion method is used for all samples.
- Ensure complete bisulfite conversion to reduce false-negative methylation results. EpiTect Bisulfite Kits ensure a conversion efficiency of 99.4–99.8%.
- Use a DNA protect mechanism to avoid DNA fragmentation and to ensure large fragment lengths sufficient for sensitive HRM experiments. All EpiTect Bisulfite Kits include the EpiTect DNA Protect mechanism, which avoids excessive degradation of DNA upon conversion.
- For bisulfite conversion of DNA from cells, tissue, and blood we recommend the EpiTect Plus LyseAll Bisulfite Kit, for bisulfite conversion directly from FFPE samples the EpiTect Plus FFPE Bisulfite Kit is recommended. Both kits integrate lysis of the sample and make genomic DNA isolation obsolete, thus increasing yield and quality of bisulfite converted DNA.
- Pre-bisulfite converted EpiTect Control DNA (methylated and unmethylated) can be used to set up DNA standards of various methylation degrees for quantification of HRM results (see figure Highly sensitive results — detection of even low percentages of methylated DNA).