The protocol for the solution hybridization ribonuclease protection-trichloroacetic acid precipitation has been described in detail in earlier report [21]. A 538-base pair (bp) fragment from the rat POMC cDNA or a 860-bp fragment from the rat dopamine D2 receptor cDNA was cloned into the polylinker region of either pSP64 or pSP65 plasmids (Promega, Madison, WI) in both the sense and antisense orientations. A 531 bp fragment from the rat orexin (or hypocretin) cDNA was cloned into the polylinker region of pBC SK+ (Stratagene, La Jolla, CA). A 502 bp fragment from the rat AVP cDNA or a 1201 bp fragment from the rat V1b receptor cDNA was cloned into the polylinker region of pCR II (Invitrogen, Carlsbad CA). The plasmid pS/E (a pSP65 derivative) was used to synthesize riboprobe for the 18S rRNA to determine total RNA. 33P-labeled cRNA antisense probes and unlabeled cRNA sense standards were synthesized using a SP6, T3 or T7 transcription system. A denaturing agarose gel containing 1.0 M formaldehyde showed that a single full-length transcript had been synthesized from each plasmid.
