Total RNA was extracted from chicken hypothalamus by Trizol reagent (ThermoFisher Scientific, Rockford, IL) according to manufacturer's recommendations, DNAse treated and reverse transcribed (Quanta Biosciences, Gaithersburg, MD). RNA integrity and quality was assessed using 1% agarose gel electrophoresis and RNA concentrations and purity were determined for each sample by Take three micro volume plate using Synergy HT multi-mode microplate reader (BioTek, Winooski, VT). The RT products (cDNAs) were amplified by real-time quantitative PCR (Applied Biosystems 7500 Real-Time PCR system) with SYBR Green Master Mix (ThermoFisher Scientific, Rockford, IL). Oligonucleotide primers used for chicken hypothalamic neuropeptide Y (NPY, Accession n° NM_205473 ), agouti-related peptide (AgRP, Accession n° AB029443 ), proopiomelanocortin (POMC, Accession n° AB019555 ), cocaine and amphetamine regulated transcript (CART, Accession n° KC249966 ), orexin (ORX, Accession n° AB056748 ), orexin receptor 1/2 (ORXR1/2, Accession n° AB110634 and NM_001024584 , respectively), leptin receptor (Ob-R, Accession n° NM_204323 ), HSP 60, 70 (HSP60, 70, Accession n° NM_001012916 and JO2579 ), heat shock factor 1-4 (HSF1-4, Accession n° L06098 , NM_001167764 , L06126 , and NM_001172374 , respectively), AMP-activated protein kinase alpha 1/2 (AMPKα1/2, Accession n° NM_001039603 and NM_001039605 , respectively), mechanistic target of rapamycin (mTOR, Accession n° XM_417614 ), p70 ribosomal protein S6 kinase 1 (S6K1, Accession n° NM_001109771 ), and ribosomal 18S (Accession n° AF173612 ) as housekeeping gene were previously published (Blankenship et al., 2015 (link); Lassiter et al., 2015 (link); Nguyen et al., 2015 (link)). Oligonucleotide primers used for chicken HSP90 (Accession n° X07265) are: forward, 5′-TGACCTTGTCAACAATCTTGGTACTAT-3′ and reverse, 5′-CCTGCAGTGCTTCCATGAAA-3′ amplifying a fragment of 68 bp. The qPCR cycling conditions were 50°C for 2 min, 95°C for 10 min followed by 40 cycles of a two-step amplification program (95°C for 15 s and 58°C for 1 min). At the end of the amplification, melting curve analysis was applied using the dissociation protocol from the Sequence Detection system to exclude contamination with unspecific PCR products. The PCR products were also confirmed by agarose gel and showed only one specific band of the predicted size. For negative controls, no RT products were used as templates in the qPCR and verified by the absence of gel-detected bands. Relative expressions of target genes were determined by the 2−ΔΔCt method (Schmittgen and Livak, 2008 (link)).
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