Myeloid Leukemia

30 Picard, Silvy, and Gabert
2. Principles of RQ-PCR
Typical PCR amplification curves have three segments: an early background,
an exponential growth phase (or a log-linear phase) and a plateau phase. The
exponential growth phase begins when sufficient product has accumulated to be
detected above background and ends when the reaction efficiency falls. During
this phase, the increase in signal corresponds directly to an increase in PCR product
and is dependent on the amount of starting material (Fig. 1A).
The general principles for quantification by RQ-PCR are identical whatever
the chemistry and instrument used. The amplification plot is visualized in a
format of ∆Rn vs cycle number, where ∆Rn is the normalized reporter signal
relative to the baseline signal established in the first few cycles of PCR. Rn
represents the fluorescent signal of the reporter dye divided by the fluorescent
signal of the reference dye (generally Rox). The presence of this dye is obligatory
for RQ-PCR on Applied Biosystems (ABI) machines but may be omitted
on other instruments. The background fluorescence is detected in all wells that
contain the PCR reaction during the first few PCR cycles. A threshold level is
then defined as a level of fluorescence above the background Rn, and is typically
a multiple of the standard deviation of the background (e.g., 10-fold for
ABI instruments). This threshold, which can be manually readjusted, defines
the threshold cycle (Ct). The Ct is the cycle number in the exponential phase of
PCR for which the fluorescence emitted by the reporter dye crosses the threshold.
It is dependent on the starting template copy number, the efficiency of
DNA amplification, the PCR system, and the type of chemistry used.
Either absolute or relative quantification methods can be employed. A standard
curve is required for absolute quantification and for one of the two possible
methods of relative quantification (see Subheading 4.). The standard
curve is constructed using 1:10 serial dilutions of a positive control (e.g., a
plasmid containing the target sequence, or DNA or cDNA from an appropriate
cell line). The Ct of these 10-fold serial dilutions vs log 10 of their initial concentrations
produces a linear curve, which is defined by several characteristics:
(1) the y-intercept, which provides the Ct value equivalent to one target copy in
the case of a plasmid standard curve (Fig. 1C), and (2) the slope, which is
indicative of the difference in Ct values equivalent to a 1-log difference in the
initial quantity of target sequence (i.e., the efficiency of amplification). Theoretically,
the slope of the standard curve should be –3.3, because for a PCR
with 100% efficiency, the Ct value decreases by one cycle when the concentration
of template is doubled. Thus the difference of one log or 10X in the initial
template concentration is equivalent to a Ct difference of 3.3 (Fig. 1B).
2.1. Chemistry
There are various kinds of fluorescence techniques used in PCR-based
detection. The most common methods use either a DNA-binding dye (nonspe-