QUANTITATIVE REVERSE-TRANSCRIPTASE PCR TO GAUGE INCREASED PROTEASE-ACTIVATED RECEPTOR-1 (PAR-1) MESSENGER-RNA 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 doma in superfamily. On neural cells, thrombin has deleterious effects, kil ling neurons through apoptosis. Consequently, knowledge of PAR-1 expre ssion in the nervous system may help to elucidate the role of thrombin in neurodegenerative disease. We developed a mimic construct to facil itate the highly sensitive technique of quantitative reverse transcrip tase to PCR (qRT-PCR) to measure the differential expression of low co py number PAR-1 mRNA in neurodegenerative model systems. In this artic le, we report our results comparing homozygous wobbler (wr/wr) mice an d 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 PAR1 mRNA in t he cervical spinal cord of wr/wr.