Quantifying Neuropeptide Y Receptor Expression in Rat Brain

The rats were euthanized with an overdose of sodium pentobarbital (200 mg/kg, i.p.), and the brain tissues were collected and cut into 500 μm coronal sections. PVN tissues were dissected by punch-out technique as previously described [20 (link)], and pieces of cerebral cortex from the same section were also obtained. Total RNA from PVN and cerebral cortex was isolated in accordance with the procedure of the RNeasy kit (Qiagen, Valencia, CA, United States). Following reverse transcription of total RNA, the mRNA quantifications for NPY, NPY Y1R, and NPY Y5R were performed through TaqMan real-time PCR method as previously described [21 (link)]. TaqMan PCR probes for rat NPY(Rn01410145_m1), NPY Y1R (Rn02769337_s1), NPY Y5R (Rn02089867_s1), and 18S rRNA (Rn03928990_g1) were purchased from Thermo Fisher Scientific (Carlsbad, CA, United States). Applied Biosystems PRISM 7500 sequence detection system was used to detect the PCR reactions. Quantitative analysis of target mRNA expression was performed with a comparative cycle of threshold (CT) fluorescence technology. The expression level of the target genes was normalized by the expression of the 18S rRNA. The arbitrary units of target mRNA were expressed as the ratio of the target mRNA concentration to the 18S rRNA of the same sample.