Quantitative reverse transcriptase PCR to gauge increased protease-activated receptor 1 (PAR-1) mRNA copy numbers in the Wobbler mutant mouse.

Thrombin acts on cells through the surface protease-activated receptor 1 (PAR-1), a G-protein-coupled member of the seven-transmembrane domain superfamily. On neural cells, thrombin has deleterious effects, killing neurons through apoptosis. Consequently, knowledge of PAR-1 expression in the nervous system may help to elucidate the role of thrombin in neurodegenerative disease. We developed a mimic construct to facilitate the highly sensitive technique of quantitative reverse transcriptase to PCR (qRT-PCR) to measure the differential expression of low copy number PAR-1 mRNA in neurodegenerative model systems. In this article, we report our results comparing homozygous wobbler (wr/wr) mice and normal littermates. By optimizing the transcription and quantitative PCR procedures to facilitate rapid copy number determination in small RNA samples, we documented a fivefold greater level of PAR-1 mRNA in the cervical spinal cord of wr/wr.