Adult human RNA samples were obtained from Clontech either as poly(A) purified RNA (hypothalamus and adipocyte) or as total RNA (cerebellum, cortex, spleen, pancreas, lung, kidney, liver, testes and total brain). The total RNAs were purified to poly-A RNA using the Micro-Poly(A)Purist kit (Ambion) according to manufacturer's instructions. We used 20 ng of poly(A) RNA in a random-primed first-strand cDNA synthesis using SuperScript II (Invitrogen) according to manufacturer's instructions. The resulting cDNAs were diluted fourfold, and 5 μl of each sample were used in 12 μl reaction with SYBR Green PCR Master Mix kit (Applied Biosystems). Quantitative PCR reactions were done in triplicate on an ABI 7900HT (Applied Biosystems). We calculated expression levels from their average crossing points and expressed relative to the control gene EEF2 (elongation factor 2) and normalized to levels of gene-specific expression in total brain.
