Techne PrimeQ - Manual (Page 126)

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3.14.6.2 The standard curve
Linear regression is used to generate a straight line, y = mx + c whereby:
• y = the slope, ideally this should be approximately -3.32.
• c = the value at which the line intercepts the y axis.
• R
2 = Correlation coefficient (mean squared error of the determination) – a perfect correlation
has a value of 1.
• E = efficiency of the reaction whereby E = 10
-1/slope
. The ideal value is 2 for 100% efficiency
(i.e. a doubling of target every cycle). A value less than 2 suggests that the PCR is sub-
optimal while above 2 suggests the amplification of non-specific products. When comparing
two reporters in one well, it is important for both PCRs to have a near equal efficiency
(typically in the range of 1.95 and 2.05).
If there is more than one reporter dye with standards (for example in Relative Quantification) then
the standard curve for each reporter can be displayed on the same graph for comparison.
This is useful for optimization of reactions in preparation for multiplexing. See section 4.11.
3.14.6.3 Calculating unknowns
The standard curve can be used to calculate the starting concentration of unknowns by reading
their Cq values from the graph. In Quansoft this is performed automatically and the calculations
are displayed in the results table.
y = -3.379x + 36.724
R² = 0.996 E = 1.977
Log Concentration
7 6 5 4 3
C
y
c
le
N
u
m
b
e
r
26
24
22
20
18
16
14