You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
Diagnosis of PML-RARA-Positive APL 123<br />
6. If necessary, increase the amount of cDNA to be amplified (3–4 µL of cDNA<br />
instead of the usual 2 µL).<br />
7. Improvement of RNA extraction and cDNA synthesis. As an alternative to the<br />
standard Chomczynski-Sacchi guanidinium thiocyanate-phenol-chloroform<br />
method, several commercial kits are now available for RNA extraction. RNA and<br />
cDNA can now be obtained from a limited number of cells either manually or<br />
automatically. For cDNA synthesis, the use of random hexamers instead of oligodT<br />
results in a better yield, even in the presence of partially degraded RNA. In<br />
our hands, the use of random nonamers as cDNA primers does not significantly<br />
increase the sensitivity of the test.<br />
8. Assessment of the efficiency of the reverse transcription reaction. Perform a PCR<br />
to amplify a properly selected control gene using the same cDNA that is being<br />
tested for the presence of PML-RARA transcripts. The selection of the control gene<br />
should take into account several factors; in particular, its rate of degradation should<br />
be similar to that of PML-RARA (17). We prefer the use of ABL to RARA as a<br />
control, because the latter is normally more stable and accordingly its expression<br />
may give a false impression regarding the integrity of the RNA that is being tested.<br />
9. Control of the quality of the RNA through RQ-PCR. Unlike RT-PCR, RQ-PCR<br />
allows monitoring of the rate of accumulation of the amplification products, the<br />
course of the reaction (real-time analysis), and the “threshold cycle” (Ct) of the<br />
chimeric and control genes. If the Ct value for the control gene is extremely high<br />
(e.g., above 29–30), this indicates poor quality of the tested sample (as a result of<br />
either RNA degradation or low efficiency of the cDNA synthesis), and accordingly<br />
it renders the sample nonevaluable (18,19).<br />
10. Participate in inter-laboratory quality control on blinded diagnostic and followup<br />
samples.<br />
11. Positive and negative controls are to be used in each PCR experiment in order to<br />
verify the efficiency of the reaction and to check for possible contamination.<br />
12. Check the test sensitivity periodically by performing PCR dilution experiments<br />
on a positive cDNA. This is mandatory any time a newly synthesized primer set<br />
is introduced.<br />
13. Perform a nested PCR analysis to increase the test sensitivity and specificity even<br />
with diagnostic samples. This strategy helps avoid false-negative results in<br />
samples that have not been analyzed immediately or have not been preserved<br />
properly.<br />
14. In follow-up samples during treatment, it is not unusual to detect the presence of<br />
amplified nonspecific products, which appear as single or multiple bands in the<br />
electrophoresis gel, even when the primers and PCR protocols are used according<br />
to the Biomed-1 Concerted Action Report (12). The problem is more frequently<br />
encountered when a bcr3 breakpoint is amplified. The nonspecific<br />
products usually differ in size from the expected band, but it may happen that the<br />
observed amplified band(s) is(are) in the same range as those seen in a positive<br />
case. Figure 3 shows some representative cases. These results should be interpreted<br />
conservatively, especially in a patient who has been persistently negative
Change language