Techne PrimeQ - Manual (Page 107)

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the early stages is expanded and the Cq value can be determined more accurately. Although
some values may be negative when plotted on a log scale (especially if the data has been
background corrected) this will not affect the accuracy of the data.
3.9.4 First derivative maximum
Simple to perform and requiring no user input, this method determines the quantification cycle by
calculating the point on the reaction curve at which the rate of change in fluorescence is fastest.
As this approach uses the shape of the curve for its calculations, it does not require the setting of a
noise threshold or crossing line. To choose this method, simply select First derivative maximum
in the Quantification Wizard and the software will automatically calculate the Cq value for each
well by taking the first derivative of the curve and then finding the maximum. This method is better
suited for high copy number templates (>1000 copies).
3.9.5 Standard curve
If standards have been defined in the plate layout, the quantification cycle for each can be plotted
against the log of its concentration to produce a standard curve. Linear regression is then used to
generate a straight line plot and the standard curve can then be used to calculate the starting
concentration of the unknowns from their Cq values.
Plotting the Cq against log concentration of
the standards generates a standard curve.
The slope of the line provides information about the efficiency (E) of the reaction whereby:
E = 10
-1/slope
A reaction of 100% efficiency would produce a value of 2 such that a doubling of an amplification
product occurs each cycle. If the value is greater than 2, it suggests amplification of non-specific
products, while lower than 2 suggests a component is limiting the reaction or that the reaction
conditions need to be optimized.
To make any comparison between samples or between two reactions in the sample well, the user
has to be confident of comparing like-for-like i.e. that the reactions are being measured at
comparable points in the amplification curve. This premise follows for any of the analysis methods,
be it quantification, dissociation curve or allelic discrimination. This allows the user to draw true
comparisons and therefore to get the most out of each assay.
3.9.6 Dissociation curve
Dissociation curve analysis can add to the information obtained from the PCR. Also known as
melting curve analysis, it measures the temperature at which the DNA strands separate into single
strands. This provides a measurement of the melting temperature or Tm, taken as the point at
which 50% of the double stranded DNA (dsDNA) molecules are dissociated.
Using an intercalating dye the dsDNA is dissociated into its separate strands by a stepwise
increase in temperature, with the fluorescence data collected at each temperature step. The PCR
products can be seen to ‘unzip’ at a specific temperature (the Tm), and as the Tm is characteristic
y = -3.402x + 43.278
R² = 0.999 E = 1.967
Log Concentration
9 8 7 6 5 4 3 2
C
y
c
le
N
u
m
b
e
r
36
34
32
30
28
26
24
22
20
18
16
14
y = -3.402x + 43.278
R² = 0.999 E = 1.967
Log Concentration
9 8 7 6 5 4 3 2
C
y
c
le
N
u
m
b
e
r
36
34
32
30
28
26
24
22
20
18
16
14