Biomedical Engineering Reference
In-Depth Information
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0.1
0.01
2
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Cycles
(b)
Figure 6.4
(continued)
value but is influenced by changing reaction conditions. Hence, if background fluorescence
varies, the value of a
C
q
recorded for any particular sample is also going to be variable.
Therefore, it is essential to understand that, on its own, a
C
q
value is meaningless; conse-
quently, quoting a
C
q
is not sufficiently informative to allow a confident assessment of any
conclusion drawn from the RT-qPCR experiment.
The
C
q
must be used to calculate a corresponding quantitative measurement, such as a
copy number. This can be done in several ways, most commonly by using a standard curve
obtained from a serially diluted standard solution of RNA or DNA to report copy numbers
relative to that standard curve ('absolute quantification') [2] or by expressing the difference
in the
C
q
values of a target RNA and a calibrator sample RNA and normalizing these as
a ratio to one or (preferably) more internal reference RNA samples (relative quantification)
[79]. Other methods for quantification of RNA levels exist, especially those based on report-
ing relative gene expression ratios based on calculating individual amplification efficiencies
for each PCR assay [80, 81], but these are not in common use, as yet.
'Absolute' quantification is not really absolute, but is usually a measure relative to a
standard curve; nevertheless, the term is in general use for this method of quantification. It
is based on the use of an external standard dilution series with a known concentration of
initial target copy number, which can be used to generate a standard curve of cycle threshold
number (
C
q
) against initial target copy number [82]. To maximize accuracy, the dilutions
are made over the range of copy numbers that include the amount of target mRNA expected
