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.

Additional recommendations:

Analyze fragments no greater than about 350 bp. Larger products can be analyzed successfully but usually with lower resolution. For SNP analysis, the recommended amplicon size is between 70 and 150 bp. This is simply because a single base variation affects the melting behavior of a 100 bp amplicon more than a 500 bp amplicon, for example.

Samples contaminated with post-amplification artifacts such as primer dimers or nonspecific products can make HRM results difficult to interpret. Use the Type-it HRM PCR Kit and the EpiTect HRM PCR Kit to generate highly specific PCR products for HRM analysis.

The capture and analysis of real-time amplification data can be extremely useful when troubleshooting HRM analyses. Amplification plots should have a CT of no more than 30 cycles. Products that amplify later than this (due to too little starting template amount or template degradation effects) typically produce variable HRM results due to amplification artifacts.

Prior to HRM, examine real-time plot data carefully for abnormal amplification curve shapes. Plots having a log-linear phase that is not steep, is jagged, or that reaches a low signal plateau compared to other reactions can indicate poor amplification or a fluorescence signal that is simply too low. Poor reactions can be caused by reaction inhibitors, too little dye, incorrect reaction setup, etc. HRM data from such samples can be inconclusive or of lower resolution.

The concentration of a DNA fragment affects its melting temperature (Tm). For this reason, sample DNA concentrations must be kept as similar as possible. When analyzing amplification products, ensure every reaction has amplified to the plateau phase. At plateau, all reactions will have amplified to a similar extent irrespective of their starting amount. Note however that "poor" reactions might not reach plateau with the same amplified quantity due to, for example, inconsistent assay setup (e.g., the primer concentration was too low).

All samples must be of equal volume and amount, and should contain the same concentration of dye. Sample purity should also be consistent. We recommend using the same DNA purification method for all samples to ensure standardization. DNA melting behavior is affected by salts in the reaction mix, so it is important that the concentration of buffer, Mg2+, and other salts is as uniform as possible in all samples. When using the Type-it HRM PCR Kit or the EpiTect HRM PCR Kit for HRM analysis, these requirements are completely fulfilled.

Capture HRM data points over about a 10°C (or greater) window, centered around the observed Tm. This provides enough baseline data points for effective curve normalization and will result in tighter replicates and easier data interpretation.

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).