Detection and quantitation by competitive RT-PCR of Epo mRNA in whole rat testis and in Sertoli and peritubular myoid cells in basal conditions and after hormonal stimuli.

(A) Quantitation of Epo mRNA transcripts in rat testis; 5 μg total RNA from rat testis with the indicated amounts of competitor Epo cRNA was reverse transcribed and amplified; no RNA (negative control), kidney total RNA (positive control). (B) Quantitation of Epo mRNA in CoCl₂-stimulated rat Sertoli cells; 10 μg total RNA from primary cultures of rat Sertoli cells, 5 μg from kidney, 10 μg from skeletal muscle, and the indicated amounts of Epo cRNA competitor were reverse transcribed and amplified. Sertoli cells were stimulated with 100 CoCl₂ for 18 hours. No Epo transcripts were detected from skeletal muscle (negative control). (C) Detection of Epo mRNA transcripts in rat Sertoli cells; 5 μg total RNA from kidney and testis and 10 μg total RNA from rat Sertoli cells with a standard amount of the competitor (50 fg) were reverse transcribed and amplified. Sertoli cells were incubated for 18 hours with 100 ng/mL FSH and 100 μM CoCl₂. (D) Detection of EPO mRNA transcripts in rat peritubular myoid cells; 10 μg total RNA from primary cultures of rat peritubular myoid cells in basal condition and stimulated with 100 μM CoCl₂ for 18 hours and 5 μg total RNA from rat kidney were reverse transcribed and amplified.