Rotor-Gene Q MDx CE User Manual 02/2022
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Comparative quantitation results.
The reaction efficiency is shown in the “Amplification” column as a score out of 2
(2 = 100% efficiency).
Note
: If you suspect the presence of primer–dimers or nonspecific products, assess reactions
by drawing a derivative plot using the
Melt
option from the
Analysis
window. Ensure that
there is a single peak, indicative of a single product. If possible, run a gel to check that there
is a single amplification product. If there is more than one product, the reaction should be
repeated or reoptimized.
10.9
HRM data analysis
HRM analysis enables both visual- and auto-calling of genotypes. Results can be viewed as either
a normalized melt plot or a difference plot. Normalized curves provide the basic representation of
the different genotypes based on curve shifting (for homozygotes) and curve shape change (for
heterozygotes).
Difference plots are an aid to visual interpretation. They plot the difference in fluorescence of a
sample to a selected control at each temperature transition. Difference plots provide an alternative
view of the differences between melt curve transitions.
Note
: First derivative melt curve analysis (as used by the standard
Melt
option in the
Analysis
window) is considered inappropriate for HRM analysis. This is because any derivation of the
data adds artificial noise and makes data interpretation more difficult.
The following steps describe the analysis of HRM results using Rotor-Gene Q software.
1.
Select the
HRM
option from the
Analysis
window.
