Expression of the Erythropoietin Receptor: RESULTS(4)

Detection of EPO-R mRNA

By loading 20 ^g of total RNA or poly(A)+ RNA from the villous placenta and from isolated first- and third-trimester trophoblast cells, we were able to detect a faint hybridization signal by Northern analysis at approximately 1.8 kilobases (data not shown). However, we used RT-PCR to verify the expression of placental EPO-R mRNA (Figs. 9 and 10). A 197-bp PCR product was amplified from reverse-transcribed RNA extracted from first- and third-trimester placentas, Jar trophoblast-derived choriocarcinoma cells, isolated and immunopurified first- and third-trimester trophoblast cells, and the positive control fetal mouse liver cells (Figs. 9A and 10A).

The 197-bp product was predicted based on the internal set of primers. Deletion of RNA or reverse transcriptase from the RT reaction yielded no demonstrable bands by ethidium bromide detection. As additional evidence that the amplified 197-bp PCR product was ultimately derived from EPO-R mRNA in these placental tissues and trophoblast cells, restriction enzyme digestion was performed using AvaII (Figs. 9B and 10B). The predicted and observed sizes of the two restriction enzyme fragments of 140 and 57 bp were identical. These were also observed for the positive control fetal mouse liver cells.
Fig8Expression of the Erythropoietin
FIG. 8. Detection of EPO-R (A) and cytokeratins (B) in immunoblots of placental homogenates from lane 1, first trimester; lane 2, second trimester; lane 3, third trimester; and lane 4, preeclamptic villous placental tissues. C) The negative control blot where the mouse IgG1 к was substituted for primary antibody. Molecular weight standards X 10~3 are indicated on the left.

Fig9Expression of the Erythropoietin
FIG. 9. A) Detection of EPO-R mRNA by nested-primer RT-PCR and visualization of the amplified 197-bp product by agarose gel electrophoresis in the presence of ethidium bromide. Lanes 1 and 7, size standard; lanes 2 and 8, RNA deleted from the RT reactions (negative control); lane 3, fetal mouse liver cells (positive control); lane 4, reverse transcriptase omitted from the RT reaction for RNA from fetal mouse liver cells (negative control); lane 5, Jar trophoblast-derived choriocarcinoma cells; lane 6, reverse transcriptase omitted from the RT reaction for RNA from Jar cells; lane 9, isolated, immunopurified first-trimester trophoblast cells; lane 10, reverse transcriptase omitted from the RT reaction for RNA from first-trimester trophoblast cells; lane 11, isolated, immunopurified third-trimester trophoblast cells; lane 12, reverse transcriptase omitted from the RT reaction for RNA from third-trimester trophoblast cells. B) Restriction enzyme digestion using AvaII and visualization of the 140- and 57-bp fragments by agarose gel electrophoresis in the presence of ethidium bromide. Lanes 1, 3, 5, and 7, size standard; lane 2, fetal mouse liver cells; lane 4, Jar trophoblast-derived choriocarcinoma cells; lane 6, isolated, immunopurified first-trimester cells; lane 7, isolated, immunopurified third-trimester cells. See Materials and Methods for details.

Fig10Expression of the Erythropoietin
FIG. 10. A) Detection of EPO-R mRNA in placental tissues by nested-primer RT-PCR and visualization of the amplified 197-bp product by agarose gel electrophoresis in the presence of ethidium bromide. Lanes 1 and 5, size standard; lanes 2 and 6, RNA deleted from the RT reactions (negative control); lane 3, first-trimester villous placenta; lane 4, reverse transcriptase omitted from the RT reaction for RNA from first-trimester villous placenta (negative control); lane 7, third-trimester villous placenta; lane 8, reverse transcriptase omitted from the RT reaction for RNA from third-trimester villous placenta. B) Restriction enzyme digestion using AvaII and visualization of the 140- and 57-bp fragments by agarose gel electrophoresis in the presence of ethidium bromide. Lanes 1 and 3, size standard; lane 2, first-trimester villous placenta; lane 4, third-trimester villous placenta.

This entry was posted in Human Placenta and tagged Erythropoietin Receptor, Human Placenta, Trophoblast Cells.