Selection of ovine housekeeping genes for normalisation by real-time RT-PCR; analysis of PrP gene expression and genetic susceptibility to scrapie

Previous to the PrP gene expression analysis the stability of several housekeeping (HK) genes was assessed in order to select the best ones for relative quantification. The normalisation of gene expression was carried out using a minimum of three HK genes in order to detect small expression differences more accurately than using a single control gene. The expression stability analysis of six HK genes showed a large tissue-associated variation reflecting the existence of tissue-specific factors. Thereby, a specific set of HK genes was required for an accurate normalisation of the PrP gene expression within each tissue. Statistical differences in the normalised PrP mRNA levels were found among the tissues, obtaining the highest expression level in obex, followed by ileum, lymph node, spleen, cerebellum and cerebrum. A tendency towards increased PrP mRNA levels and genetic susceptibility was observed in central nervous system. However, the results did not support the hypothesis that PrP mRNA levels vary between genotypes.The results on PrP gene expression presented here provide valuable baseline data for future studies on scrapie pathogenesis. On the other hand, the results on stability data of several HK genes reported in this study could prove very useful in other gene expression studies carried out in these relevant ovine tissues.Scrapie is a neurodegenerative disease of the group of transmissible spongiform encephalopathies (TSEs) that affects sheep and goats [1,2]. The lesions appear mainly in the nervous system in the form of vacuoles triggered by the conversion of the cellular prion protein (PrPc) into the abnormal isoform (PrPSc) followed by its pathological accumulation [2]. Although the exact origin of the disease remains unknown, the "protein only" hypothesis supports the concept that PrPSc is the transmissible agent causing the disease [2]. Therefore, the presence of PrPc is essential to develop scrapie [3,4]. However, little is known about the physiologica