Exp Clin Endocrinol Diabetes 2003; 111 - P40
DOI: 10.1055/s-2003-817582

Combining a punch technique with quantitative real-time PCR to measure exact amounts of orexin receptor mRNA in specified brain nuclei

P Herlyn 1, O Jöhren 1, P Dominiak 1
  • 1Institute of Experimental and Clinical Pharmacology and Toxicology, Luebeck, Germany

Orexins are considered to be important in sleep, feeding and energy homeostasis. Their two G protein-coupled receptors (OX1 and OX2) are expressed in many areas of the brain involved in the regulation of these processes. To exactly quantify the amount of orexin receptor mRNA in specific brain nuclei we have combined quantitative real-time PCR with the micro-punch technique. Male rats were decapitated, brains removed, rapidly frozen and stored at –80°C. Punches of the locus coeruleus (LC), nucleus paraventricularis (PVN), lateral hypothalamus (LH), taenia tecta (TT), raphe obscurus (RO), cortex, cerebellum, and the pineal gland (Epi) were collected from 300µm slices and homogenized in guanidinium isothiocyanate buffer. After extraction, total RNA was reverse transcribed and cDNA was amplified using OX1 and OX2 receptor specific primers by real-time PCR. In general, OX1 receptor mRNA levels were higher than those of OX2 receptor mRNA. Only in the PVN and the cerebellum, OX2 receptor mRNA was slightly higher expressed than OX1 receptor mRNA. The LC showed the highest levels of OX1 receptor mRNA (60-fold higher compared to OX1 receptor in the cortex). In descending order, TT, LH, RO, pineal gland and PVN yielded moderate levels of OX1 receptor mRNA, while the lowest amounts of OX1 receptor mRNA were found in the cortex and the cerebellum. The OX2 receptor mRNA was more evenly distributed among the examined nuclei, the highest levels appeared in the PVN, the lowest in the epiphysis. Combining the micro-punch technique with quantitative reverse transcription real-time PCR provides an adequate method for the determination of exact amounts of mRNA levels in distinct brain areas. We were able to quantify mRNA levels of OX1 and OX2 receptors in brain punches as small as 0.064mm3. Thus Our data extend previous results yielding a rather roughly estimated distribution of orexin receptors by semi-quantitative methods, such as in situ hybridisation.