Myeloid Leukemia

Diagnosis of PML-RARA-Positive APL 121
3.3. First-Round and Nested PCR Conditions (see Notes 10–13)
1. Initial DNA melting denaturation and Taq polymerase activation: 95°C for 2 min
(the time depends on the kind of polymerase that is used).
2. PCR cycles: 94°C for 30 s (melting), 65°C for 30 s (annealing), 72°C for 30 s
(extension), for a total of 35 cycles. No final extension is needed.
3.4. Gel Electrophoresis
1. A 1% gel is made by boiling 1 g agarose in 100 mL 1X TBE.
2. Cool to approx 50°C and add 2 µL of ethidium bromide; NB: ethidium bromide
is carcinogenic; always wear gloves!
3. Pour the agarose into an appropriate casting tray.
4. At the end of PCR, electrophorese 10 µL of reaction product plus 2 µL of loading
buffer for 1 h at 80–90 volts.
5. Examine the results by ultraviolet lamp transilluminator. In positive samples from
patients at diagnosis, single bands are usually visualized in first-round PCR analysis.
However, multiple PCR bands, caused by alternative splicing of PML exons,
can appear in bcr1/2-positive samples when amplified using primers for bcr3
breakpoint identification (13) (see Note 14).
4. Notes
1. The sensitivity of PML-RARA fusion gene detection by RT-PCR is lower in comparison
with the detection of other fusion genes, such as BCR-ABL. It is likely
that such a difference is due either to different levels of expression or to greater
instability of the PML-RARA fusion transcripts. PML-RARA mRNA undergoes
rapid degradation. We have observed a one-third to one-half log reduction for
every 24 h that a BM sample is left at room temperature, as shown in Fig. 2. This
problem is usually not relevant at diagnosis, but can be crucial for the assessment
of MRD during follow-up. As a consequence, analysis can result in a false-negative
result, or in the appearance of faint bands in agarose gel electrophoresis despite
the presence of appropriate bands for the control genes (ABL or RARA) that
are normally used to assess the quality of the cDNA. Accordingly, the quality of
RNA is an essential step in the procedure. Notes 2–14 reflect several years’ experience
as the referral diagnostic laboratory in Italy for both adult and childhood
APL patients (9).
2. Analysis of BM samples. Owing to the several critical parameters that we listed,
and to the relatively low sensitivity of the RT-PCR, diagnosis and monitoring of
BM samples must be preferred to PB, where the blast cells count could be low.
3. Rapid processing of the bone marrow (BM) sample. Perform Ficoll-Paque density
gradient centrifugation to obtain mononuclear cells (MNC); lyse the sample
immediately in GTC, RNAzol, or Trizol; and freeze at –20°C or –70°C. When
processed in this way, the sample can be preserved indefinitely. The use of dry
pellets of MNC without lysing solution has been suggested by some groups. How-