In addition to identification of EPO-R protein, we also detected EPO-R mRNA in placental tissues and trophoblast cells using RT-PCR. Levels of EPO-R message do not seem to be very abundant, as others have reported its detection by Northern analysis in positive control TF-1 cells when 10 ^g of poly(A)+ RNA was used. We noted only faint hybridization signals in placental villous tissues and isolated first- and third-trimester trophoblast cells.
To improve both the sensitivity and specificity of the assay, we used nested-primer RT-PCR, which revealed an intense, solitary product of 197 bp by agarose gel electrophoresis and eth-idium bromide staining (Figs. 9 and 10). Using restriction enzyme digestion, we verified that the 197-bp amplicon was derived from EPO-R mRNA in the placental tissues and trophoblast cells, since the predicted and observed sizes of the two fragments generated by incubation with AvaII, 140 and 57 bp, were identical. The nested-primer PCR approach that we employed did not permit us to determine whether alternative splice forms of EPO-R mRNA consistent with the soluble or truncated EPO-R are present in placental tissues and trophoblast cells because the internal primers did not span the insertion sites for these alternative species. Finally, we cannot completely exclude the possibility that the EPO-R mRNA faintly detected on Northern analysis and robustly by RT-PCR was derived solely from contaminating cells in the immunopurified trophoblast preparation (< 3%). However, the presence of mRNA in the Jar tro-phoblast-derived choriocarcinoma cell line, as well as protein expression by trophoblast cells in placental tissues on immunocytochemistry, makes this possibility an unlikely one.