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266 Martini, Teofili, and Larocca<br />
ried out Northern blot analysis on total RNA extracted from granulocytes and<br />
PRV-1 overexpression was detected by using a radio-labeled probe specific for<br />
the PRV-1 gene. Since the PRV-1 gene cloning, several studies have been published<br />
investigating PRV-1 expression across the various CMD, in an attempt<br />
to distinguish them from one another and from secondary erythrocytosis and<br />
thrombocytosis (10–18). In most of these studies, the expression of PRV-1<br />
mRNA was quantified by using a real-time polymerase chain reaction (PCR)<br />
analysis (11–18). In contrast, we assessed the expression of PRV-1 gene<br />
through a qualitative approach by using a specific reverse transcriptase PCR<br />
(10). Our method requires the crucial isolation of a 95% pure granulocyte population,<br />
RNA extraction and DNase treatment, phenol/chlorophorm purification,<br />
and then reverse transcription and a specific PCR. PCR products are separated<br />
on agarose gels and, after staining with ethidium bromide, the results are visualized<br />
under ultraviolet (UV) light. This method represents a rapid and reliable<br />
procedure for discriminating patients with PV and ET from patients affected<br />
by secondary erythrocytosis and thrombocytosis.<br />
2. Materials<br />
1. Phosphate-buffered saline (PBS): 0.2 g KCl, 8 g NaCl, 0.2 g KH2PO4, 1.15 g<br />
Na2HPO4 (pH 7.4).<br />
2. Hespan: 6% hetastarch in 0.9% NaCl (DuPont Pharma, Willington DE).<br />
3. Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden).<br />
4. TRIZOL reagent, stored at 4°C (Invitrogen, Carlsbad, CA).<br />
5. Chloroform.<br />
6. Isopropyl alcohol.<br />
7. 75% ethanol (in diethylpyrocarbonate [DEPC]-treated water).<br />
8. RNase-free water (treated with 0.01% DEPC).<br />
9. Deoxyribonuclease I, amplification grade (Invitrogen).<br />
10. 10X DNase I reaction buffer: 200 mM Tris-HCl (pH 8.4), 20 mM MgCl2, 500 mM KCl.<br />
11. 25 mM ethylenediamine tetraacetic acid (EDTA), pH. 8.0.<br />
12. Phenol/chloroform/isoamyl alcohol (25:24:1).<br />
13. Ammonium acetate (7.5 M).<br />
14. Ethanol (100%).<br />
15. Oligo(dT) 12–18 (0.5 µg/µL).<br />
16. 10X reverse-transcription (RT) buffer: 200 mM Tris-HCl (pH 8.4), 500 mM KCl.<br />
17. 25 mM MgCl2. 18. 0.1 M dithiothreitol (DTT).<br />
19. 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP).<br />
20. SuperScript II RT (50 U/µL) (Invitrogen).<br />
21. RNaseOUT Recombinant Ribonuclease Inhibitor (40 U/µL).<br />
22. Escherichia coli RNase H (2 U/µL).<br />
23. Taq DNA Polymerase in Storage Buffer A (5 U/µL) (Promega, Madison WI).
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