Tichopad Ales, Pfaffl Michael W, Didier Andrea
Institute of Physiology, FML Weihenstephan, Technical University of Munich, Weihenstephaner Berg 3, 85354 Freising, Germany.
Mol Cell Probes. 2003 Feb;17(1):5-10. doi: 10.1016/s0890-8508(02)00114-7.
In recent studies PrP mRNA was determined mostly by in situ hybridisation or Northern Blot analysis--methods not suitable for absolute quantification of mRNA copy numbers. Herein we report on bovine prion mRNA quantification using calibrated highly sensitive externally standardized real-time RT-PCR with LightCycler instrument. Total RNA was isolated from nine different regions of the CNS and seven peripheral organs. PrP(c) mRNA copy numbers could be determined in all tissues under study. In approval with prior studies high mRNA level was found in Neocortex and Cerebellum. Lymphatic organs showed at least as high expression levels of prion mRNA as overall brain. Lowest expression was detected in kidney. Results of our study provide insight into the involvement of different organs in pathogenesis with respect to prion mRNA expression. LightCycler technology is currently considered the most precise method for nucleic acid quantification and showed to be powerful tool for further studies on prion diseases pathogenesis.
在最近的研究中，朊蛋白（PrP）mRNA大多通过原位杂交或Northern印迹分析来测定——这些方法不适用于mRNA拷贝数的绝对定量。在此，我们报告使用校准的高灵敏度外部标准化实时逆转录聚合酶链反应（RT-PCR）和LightCycler仪器对牛朊病毒mRNA进行定量。从中枢神经系统的九个不同区域和七个外周器官中分离出总RNA。在所研究的所有组织中都可以测定PrP（c）mRNA拷贝数。与先前的研究一致，在新皮质和小脑中发现了高mRNA水平。淋巴器官中朊病毒mRNA的表达水平至少与整个大脑一样高。在肾脏中检测到最低表达。我们的研究结果为不同器官在朊病毒mRNA表达方面参与发病机制提供了见解。LightCycler技术目前被认为是核酸定量最精确的方法，并且被证明是进一步研究朊病毒疾病发病机制的有力工具。
