ABSTRACT
The hypothalamus plays a central role in the integrated control of feeding, energy homeostasis, circadian rhythms, sex behavior, and arousal. Directional tag PCR subtractive hybridization method was used to identify mRNAs selectively expressed in the hypothalamus. This technology allows comparison of two populations of mRNAs, named target and driver, and isolation of target-specific mRNAs. Briefly, cDNA libraries are prepared from driver and target tissue, and cloned in different plasmid vectors. The target cDNA library is then converted into single stranded cDNA and hybridized with a 50-fold molar excess of in vitro transcribed RNA obtained from the driver library. The hybridization conditions allow rapid annealing of common sequences, and target-specific sequences remain as single stranded cDNAs, which is separated from double stranded cDNA:RNA hybrids by hydroxylapatite chromatography, amplified by PCR and cloned (1). This method has proved successful to identify striatal-enriched cDNAs (1), and to isolate preprocortistatin, a neuropeptide related to somatostatin involved in cortical synchronization and sleep (2).
