Abnormally high gene mRNA expression in PV erythroid progenitors.

Highly purified day-8 ECFCs were generated from 6 healthy donors and 6 patients with PV. Total RNAs were isolated and 20 μg of RNA were analyzed for the presence of RNA transcripts for p130, Rb, p107, p53, p57, p21, p19, p18, p16, p14/15 related to cell proliferation, using the hCC-2 Multi-Probe Template Set (Pharmingen). L32 andGAPDH were included as internal controls. RNase protection assays (RPAs) were performed with the MAXIscript and RPA II Ribonuclease Protection Assay Kits (Ambion) according to the manufacturer's protocols. Protected transcripts were separated by denaturing polyacrylamide gels and quantified by autoradiography. The band with a strikingly higher expression is indicated by an arrow.