Biomedical Engineering Reference
In-Depth Information
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32
30
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20
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14
1.00 e
+
03
1.00 e
+
04
1.00 e
05
Initial Quantity (copies)
+
1.00 e
+
06
1.00 e
+
07
1.00 e
+
08
(a)
Figure 6.4
(a) Typical standard curve used to quantitate target mRNA from colonic biopsies.
All the
C
q
quantification data from the test samples (blue triangles) in the upper picture
are contained within the dynamic range of the standard curve, which is demarcated by the
two outermost points of the standard derived from samples of a defined concentration and
represented by red squares. This allows accurate quantification of the corresponding mRNAs. (b)
Typical amplification plot obtained using a SYBR Green I assay. A single transcript has been
quantified in a number of test samples and a serial dilution of standard material using SYBR Green
I as the reporter. The two replicates for the three most concentrated standard samples (traces
on left of the graph colored blue, red and green) illustrate a good standard of pipetting. The
slopes of all the amplification plots are identical, indicating that the amplification efficiencies of
every sample are the same. The high relative fluorescence (
R
n
) value is typical of SYBR Green I
assays. (See Plate 6.4.)
incorporated an algorithm into their data analysis software that allows the user to determine
the efficiency of the PCR in each individual tube based on the fluorescence history of each
reaction. The software uses a second derivative of the raw amplification data to determine
the 'takeoff' point of a reaction, and the slope of the line from the takeoff point until expo-
nential amplification ends is used to calculate the amplification efficiency. This value is then
used when calculating the relative quantity of target in sample and calibrator reactions (see
Figure 6.4). At present, this approach is most useful when using SYBR Green I dye as the
reporter, because this gives a higher quantum yield per cycle, which is required to estimate
the range of logarithmic amplification.
The common measure of all RT-qPCR assays, regardless of how the sample was obtained,
reverse transcribed, or how the amplicon was detected is the
C
q
(
C
t
or
C
p
). The C
q
is
defined as the cycle when sample fluorescence exceeds a chosen threshold above calculated
background fluorescence. Different systems may refer to the generic term
C
q
using alterna-
tive terminology, such as
C
t
or
C
p
. Background fluorescence is not a constant or absolute
