The quantification of prion gene expression in sheep using real-time RT-PCR.

摘要

Determination of the transcription level of cellular prion protein (PrP(C)) is essential for understanding its poorly explained role in organisms. Scrapie in sheep is the prototype of all prion diseases. However, the expression of prion protein (PrP) mRNA in sheep has not been quantified in great detail. Herein we report on measurement of sheep PrP mRNA using absolute quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR). Total RNA was isolated from seven different regions of the central nervous system (CNS) and six peripheral organs of 18 sheep and PrP mRNA was quantified by real-time RT-PCR using an externally calibrated standard curve constructed with the recombinant PrP plasmid. The results showed that high levels of PrP mRNA were expressed in all seven regions of the brain examined, with obex and neocortex expressing the highest PrP, followed by cerebellum, spinal cord, hippocampi, conarium and thalamus, In peripheral organs examined, lymph node showed a level of PrP expression similar to that in overall brain, whereas spleen, heart, liver and lung showed moderate level of expression and kidney showed the lowest expression. Our study provided the first quantitative, tissue-specific data of PrP mRNA expression in sheep for further studies of pathogenesis of prion diseases.