Total RNA was prepared from the NAc, CPu, BLA, and hypothalamus (Hypo) by using an RNeasy mini kit (Qiagen) or TRIzol reagent (Invitrogen). First-strand cDNA synthesis was performed by using a ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). Total RNA (250 ng) was subjected to quantitative real-time PCR performed with a SYBR Fast qPCR Kit (KAPA Biosystems, Wilmington, MA, USA) on a Light Cycler PCR platform (Roche Diagnostics, Basel, Switzerland). Primers used for qPCR are listed in Table 1 . PCR was performed by using the following protocol: 95 °C for 3 min, followed by 45 cycles in total at 95 °C for 10 s and 60 °C for 30 s, then 72 °C for 1 s. Generation of specific PCR products was confirmed by melting curve analysis and DNA gel electrophoresis. Data were analyzed by using the ΔΔCt method, with normalization against GAPDH mRNA expression.Table 1
Primers used in the present study.
| Genes (Genbank Accession number) | Primers | Product length (bp) | |
|---|---|---|---|
| NMU ( | Forward | 5′-GTCCTCTGTTGTGCATCCGTT-3′ | 130 |
| Reverse | 5′-GCGTGGCCTGAATAAAAAGTA-3′ | ||
| NMUR1 ( | Forward | 5′-CGTCATCCTGCGCAACAAG-3′ | 223 |
| Reverse | 5′-CACACTCAGGGCTGTGACAT-3′ | ||
| NMUR2 ( | Forward | 5′-TGTCACCACGGTTAGCATTGA-3′ | 218 |
| Reverse | 5′-GTTTGGTGACTGTGCAGGTG-3′ | ||
| GAPDH ( | Forward | 5′-CGTCCCGTAGACAAAATGGT-3′ | 177 |
| Reverse | 5′-GAATTTGCCGTGAGTGGAGT-3′ | ||