Myeloid Leukemia

270 Martini, Teofili, and Larocca
Fig. 1. PRV-1 overexpression in essential thrombocythemia (ET) and polycythemia
rubra vera (PV). (A) PRV-1 mRNA in patients with ET (lanes 1–5) and PV (lanes 6–
10). Negative control (water) and positive control (previously tested and sequenced
cDNA) are indicated with – and +, respectively. (B) β-actin expression indicates cDNA
integrity in the same samples. MW indicates molecular-weight marker.
at 95°C for 40 s, 54°C for 40 s, 72°C for 40 s, and a final cycle of 3 min at 72°C.
3. Separate the mixture on a 2% agarose gel in 1X TBE buffer.
4. After staining with ethidium bromide, visualize the PCR products under UV light
(Fig. 1) (see Notes 3–6).
4. Notes
1. Purification of peripheral blood granulocytes is a crucial step in this method. In
fact, several studies show that PRV-1 is absent in normal mature peripheral blood
granulocytes, whereas it is expressed in promyelocytes and monocytes. Peripheral
blood mononuclear cells contain 2–6% of monocytes, and it is therefore essential
to use our protocol, which assures a 95% or greater granulocyte purification, in
order to avoid false-positive cases. The purity of the granulocyte population should
be assessed by microscope examination of Wright-Giemsa-stained slides.
2. Amplification of the β-actin gene (or other housekeeping genes) constitutes an
important step in this method. In fact, using this test it is possible verify the
presence of cDNA after the reverse transcriptase step, and then eliminate samples
without cDNA. In this control amplification, we use both an internal negative
control (water) and a positive control (previously tested cDNA).
3. Sometimes after PCR for PRV-1 amplification, we have detected a nonspecific
band on the gel. This band (approx 450 bp long) is due to DNA contamination in
the sample resulting from the failure of DNase treatment (Fig. 2). In this case, it
is necessary to repeat the DNase treatment. Moreover, the first time this assay is
employed, it is advisable to sequence the PCR product to guarantee that the
sequence of the amplified product matches the sequence of PRV-1 cDNA,
reported in GenBank (accession number AF146747). We always utilize negative
(water) and positive (previously tested and sequenced cDNA) controls in this