Rotor-Gene Q MDx CE User Manual 02/2022
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10.5
Guidelines for successful HRM analysis
The success of HRM analysis depends largely on the particular sequence under investigation.
Certain sequence motifs, such as hairpin loops or other secondary structures, localized regions of
unusually high or low GC content, or repeat sequences can all affect the outcome. In addition, use
of standardized kits and optimized protocols from QIAGEN can overcome many of the potential
challenges listed. Some simple guidelines to help ensure success are detailed below.
Analyze small DNA fragments
Analyze fragments no greater than about 250 bp. Larger products can be analyzed successfully
but usually provide lower resolution. This is because, for example, a single base variation has a
greater effect on the melting behavior of a 100 bp amplicon than on a 500 bp amplicon.
Ensure that PCR contains only specific product
Samples contaminated with post-PCR artifacts such as primer dimers or nonspecific products can
make HRM results difficult to interpret. Kits from QIAGEN for HRM analysis ensure maximum
specificity with no need for optimization.
Use sufficient preamplification template
Analysis of real-time PCR data can be very useful when troubleshooting HRM analyses.
Amplification plots should have a C
T
(threshold cycle) of less than or equal to 30 cycles. Products
that amplify later than this (due to low starting template amount or template degradation) typically
produce variable HRM results due to PCR artifacts.
Normalize template concentration
The amount of template added to the reaction should be consistent. Normalize the starting
concentrations so that all amplification plots are within 3 C
T
values of each other. This ensures input
concentrations are within a 10-fold range.
Check for aberrant amplification plots
Prior to running HRM, examine amplification plot data carefully for abnormal amplification plot
shape. Plots with 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 too
low (e.g., this could occur if primer concentration was too low). Poor reactions can be caused by