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154 Tobal and Yin<br />
two strategies for the quantification of a given transcript: competitive RT-PCR<br />
and RQ-PCR.<br />
3.4.1. Control Gene<br />
Choosing the right control gene is important for both qualitative and quantitative<br />
RT-PCR. However, a suitable control gene for qualitative RT-PCR may<br />
not necessarily be suitable for the quantitative protocol. Control genes for quantitative<br />
RT-PCR must meet two major requirements: (1) transcript levels of the<br />
control gene must not be affected by the disease in question and (2) degradation<br />
rate of the control gene must be equal to that of the gene of interest (10).<br />
Our investigations have shown that ABL is a suitable control gene for qualitative<br />
as well as quantitative RT-PCR for most AML fusion genes.<br />
3.4.2. Competitive RT-PCR<br />
Competitive PCR is suitable for the accurate quantification of genes or their<br />
transcripts, especially when real-time equipment is not available (11). In comparison<br />
to real-time quantification, competitive PCR could be seen as laborious<br />
and time consuming. The principle of the protocol is to simultaneously<br />
amplify two targets in the same reaction using the same set of primers. The two<br />
amplicons are the target gene itself in the test sample, and another DNA fragment,<br />
the competitor, which is typically a recombinant variation of the target<br />
gene that is characterized by a deletion or an insertion to produce a differentsized<br />
PCR product (Fig. 2). The number of copies in the original stock solution<br />
of the competitor is estimated by spectrophotometry. Dilutions of this competitor<br />
are made in a range of 1–10 10 molecules/2 µL, with a dilution at every<br />
order of magnitude on a logarithmic scale. The lane in which the competitor<br />
and target PCR products are present at equal amounts (the equivalence point)<br />
indicates the number of copies in the test sample.<br />
3.4.3. Real-Time Quantitative RT-PCR<br />
Unlike qualitative PCR, in RQ-PCR a fluorogenic probe is positioned and<br />
hybridized between the forward and reverse primers. The probe is labeled on<br />
the 5� with a fluorogenic reporter and on the 3� with a quencher. During the<br />
extension phase of the PCR cycle, the 5�→3� exonuclease activity of Taq polymerase<br />
cleaves the hybridized probe, resulting in the release of the fluorogenic<br />
reporter (Fig. 3). This causes an increase in the fluorescence emission of the<br />
reporter dye that is proportional to the amount of PCR product accumulated.<br />
Under appropriate conditions, this increase in fluorescence signal is also proportional<br />
to the amount of template used. The level of normalized reporter<br />
signal (∆Rn) increases during PCR as the target is amplified, until the reaction<br />
reaches a plateau. At the end of PCR amplification, real-time data analysis is<br />
performed.
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