The prerequisite for a PCR to be us

The prerequisite for a PCR to be used for quantification
is that PCR product is measured within the exponential
phase of the PCR reaction, where the amount of
amplified target is directly proportional to the input
amount of target. Therefore, quantification of gene expression
by semi-quantitative RT-PCR must be carried
out during the exponential (log) phase of the PCR reaction
and plateau phase must be avoided. Exponential
phase of the PCR reaction can be determined empirically
by amplifying equivalent amounts of cDNA over different
number of PCR cycles or by amplifying dilutions
of cDNA over the same number of PCR cycles (4,5). In
this work we performed kinetic analysis by amplifying
PCR products over different number of PCR cycles and
showed that PCR reaction was exponential between cycles
25 and 30 (Figs. 1A and C). Subsequently, »fine tuned
« kinetic analysis was performed within the given
range of cycles (Figs. 1B and D). Similar kinetic analysis
was also done for APN and another housekeeping gene
RPLP0 (data not shown). Results of semi-quantitative
RT-PCR using comparative kinetic analysis are shown
in Fig. 2. Two samples to be compared, obtained from
control and IFN- treated cells, were amplified for consecutive
cycles within the exponential phase of PCR reaction.
The optimal numbers of PCR cycles determined
were 25–28, 25–28 and 20–23 for ABL, APN and RPLP0,
respectively (Fig. 2A). The number of cDNA molecules
for ABL, RPLP0 and APN in control and IFN- treated
samples was derived from the intercept of the regression
line with the Y-axis. At this intercept, the difference
between the log of cDNA level is directly proportional
to the difference of starting level of cDNA in control
and IFN- treated samples (Fig. 2B). Finally, Fig. 2C
summarizes the results obtained by semi-quantitative
RT-PCR. Treatment of HL-60 cells with IFN- (6 ng/mL)
for 72 h upregulated expression of APN mRNA for
approximately twofold, while the expression of both
housekeeping genes, ABL and RPLP0, was unchanged.