Bio-Rad Precision Melt Analysis™ Software - Manual (Page 8)

Precision Melt Analysis Software Instruction Manual
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3. Use sufficient pre-amplification template
Analyzing real-time PCR amplification data can be extremely useful when troubleshooting
HRM analyses. Samples should have a quantification threshold (C
q
) less than 30 cycles.
Products that amplify late due to too little starting template amount or template degradation
produce variable HRM results.
4. 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 Cqs of each other, a 10-fold range.
5. Check for aberrant amplification plots
Examine amplification data carefully for abnormal amplification curve shapes. A curve with a
jagged log-linear phase or one that reaches a low signal plateau compared to other reactions
can indicate poor amplification or a fluorescence signal too low for analysis. Unsuccessful
amplification can be caused by reaction inhibitors, too little dye, or incorrect reaction set-up.
HRM analysis from such samples can cause low resolution or poor grouping.
6. Keep post-amplification sample concentrations similar
Minimizing reaction to reaction variability is critical, and using the same sample preparation
procedure will minimize this variability. Since the concentration of a DNA fragment affects its
melting temperature, ensure every reaction has amplified to the plateau phase. Poor reactions
might not reach plateau with the same amplified quantity due to inconsistent assay set-up.
7. Ensure sample-to-sample uniformity
Samples must be of equal volume and with the same concentration of dye. DNA melting
behavior is affected by salts in the reaction mix, so the concentration of buffer, Mg
2+
and other
salts should be as uniform as possible in all samples.
8. Allow sufficient data collection for pre-and post-melt regions
For easier data interpretation and results with tighter replicates, ensure enough baseline data
points were collected. This can be easily accomplished by capturing HRM data points over at
least a 10°C (or greater) window, centered around the observed T
m
of the amplified product.
Dye Compatibility
Third generation intercalating dyes, such as EvaGreen, LCGreen and SYTO 9, have been used
successfully for high resolution melt analysis. These dyes have low toxicity and can be used at
higher concentrations in real-time PCR reactions. These dyes are used at higher concentration
for greater saturation of dsDNA and less dynamic dye redistribution to non-denatured regions
of the nucleic strand during melting. The high fidelity of these third generation dyes provides
greater sensitivity and higher resolution melt profiles.
WARNING! SsoFast EvaGreen supermix cannot be used with bisulfite-converted
DNA for methylation studies. For this purpose, use Precision melt supermix.
HRM Analysis in the Literature
Review the following references to learn more about HRM analysis.
Basics of the Technology