Primers were designed based on cloned sequences for cGnRH-II (GenBank
accession # U30386, Yu et al., 1998), EF1α (GenBank accession # AB056104), and
preproorexin (GenBank accession # DQ923590) using Perlprimer software (perlprimer.
sourceforge.net) (Table 1). For all primer pairs, at least one primer lies across an exon/
exon boundary, so that primer pairs only amplify cDNA and not genomic DNA. The
primers were designed to have similar melting temperatures and to give similar
amplicon sizes. Amplified products were electrophoresed on 1.5% agarose gels and a
single fragment was obtained. Later experiments showed that these amplicons also
exhibited a single peak by dissociation curve analysis. EF1α was used as a reference
gene in this method, as it is considered a housekeeping gene and its expression did not
differ either among brain regions or among treatments (as seen by similar Ct values).