*2.11. Effect of 24-h CM vs. GM Consumption on Feeding-Related Gene Expression in the Brain Circuit*

In order to assess the effect of 24-h intake of GM and CM solutions on the expression of feeding-related genes in the brain, mice were given CM or GM (at room temperature) as the only tastant (starting at 10:00). Animals given water served as baseline controls. At 10:00 on the subsequent day (thus, 24 h after milk presentation), the animals were sacrificed via cervical dislocation. Brains were dissected out and the hypothalamus, nucleus accumbens, and brain stem excised and stored in RNAlater at −80 ◦ C until further processing. This experiment was conducted in a separate cohort of animals.

Tissues were homogenized in Trizol (Ambien), 1 mL per 0.1 g tissue. 0.2 mL chloroform was added and samples were centrifuged at room temperature for 10 min at 10,000× *g*. The clear phase containing RNA was isolated and 0.5 mL of isopropanol was added. RNA was precipitated in an ice bath for 10 min then centrifuged at 4 ◦C for 20 min at 10,000× *g*. Aqueous phase was removed from the pellets, which were then resuspended in 0.3 mL of ethanol and centrifuged at 4 ◦C for 10 min at 10,000× *g*. Liquid was removed and pellets were air-dried.

Pellets were dissolved in 8 μL DEPC water and 1 μL DNAse buffer (dNature). Samples were then incubated with 1 μL DNAse (dNature) at 37 ◦C for 30 min. DNAse was inactivated via addition of stop buffer (dNature) and incubation at 67 ◦C for 10 min. Removal of DNA was confirmed via PCR using HOT FIREPol Blend Master Mix (dNature), followed with agarose gel electrophoresis. Concentrations of RNA were measured with a nanodrop.

cDNA was synthesized from RNA samples with iScript Advanced cDNA synthesis kit (BioRad). Synthesis of cDNA was confirmed with PCR followed by agarose gel electrophoresis.

Quantitative RT-PCR (qPCR) was used to determine relative expression levels of housekeeping genes (ActB, GAPDH, β-tubulin) and of genes of interest. Reactions contained 4 μL of 25 ng/μL sample cDNA, 1 μL of each forward and reverse primers (5 μM), 10 μL iTaq Universal SYBR Green Supermix (BioRad) and 4 μL MilliQ water. qPCR experiments were performed in duplicates alongside negative controls of MilliQ water for each primer pair. Amplification protocol was initiated at 95 ◦C for 15 min, followed by 45 cycles of 15 s at 95 ◦C, 15 s at the primer-specific annealing temperature and 30 s at 72 ◦C. Primers used: GADPH F: 5 -AAGGTCATCCCAGAGC TGAA-3 , R: 5 -CTGCTTCACCACCTTCTTGA-3 ; ActB F: 5 -AGTGTGACGTTGACATCCGT-3 , R: 5 -TGCTAGGAGCCAGAGCAGTA-3 ; POMC F: 5 -GACACTGGCTGCTCTCCAG-3 , R: 5 -AGCAGCCTCCCGAGACA-3 ; Agrp F: 5 -GGATCTGTTGCAGGAGGCTCAG-3 , R: 5 -TGAAGAAGCGGCAGTAGCACGT-3 ; NPY F: 5 -GGTCTTCAAGCCGAGTTCTG-3 , R: 5 -AACCTCATCACCAGGCAGAG-3 ; MC4R F: 5 -CTTATGATGATCCCAACCCG-3 , R: 5 -GTAGCTCCTTGCTTGCATCC-3 ; β-tubulin F: 5 -CGGAAGGAGGCGGAGAGC-3 , R: 5 -AGGGTGCCCATGCCAGAGC-3 ; GHSR F: 5 -TCCGATCTGCTCATCTTCCT-3 , R: 5 - GGAAGCAGATGGCGAAGTAG-3 ; ORX F: 5 -GCCGTCTCTACGAACTGTTGC-3 , R: 5 -CGCTTTCCCAGAGTCAGGATA-3 ; OXT F: 5 -CCTACAGCGGATCTCAGACTG-3 , R: 5 -TCAGAGCCAGTAAGCCAAGCA-3 ; OXTR F: 5 -TCTTCTTCGTGCAGATGTGG-3 , R: 5 -CCTTCAGGTACCGAGCAGAG-3 ; PENK F: 5 -CGACATCAATTTCCTGGCGT-3 , R: 5 -AGATCCTTGCAGGTCTCCCA-3 ; DYN F: 5 -GACAGGAGAGGAAGCAGA-3 , R: 5 -AGCAGCACACAAGTCACC-3 ; GHRL F: 5 -CCATCTGCAGTTTGCTGCTA-3 , R: 5 -GCTTGTCCTCTGTCCTCTGG-3 ; MOR F: 5 -CCTGCCGCTCTTCTCTGG-3 , R: 5 -CGGACTCGGTAGGCTGTAAC-3 ; KOR F: 5 -CACCTTGCTGATCCCAAAC-3 , R: 5 -TTCCCAAGTCACCGTCAG-3 ; PNOC F: 5 -AGCACCTGAAGAGAATGCCG-3 , R: 5 -CATCTCGCACTTGCACCAAG-3 ; ORLP F: 5 -ATGACTAGGCGTGGACCTGC-3 , R: 5 - GATGGGCTCTGTGGACTGACA-3 ; GLP1R F: 5 -ATGGCCAGCACCCCAAGCCTCC-3 , R: 5 -TCAGCTGTAGGAACTCTGG-3 ; Cx36 F: 5 -CCAGTAAGGAGACAGAACCAGAT-3 , R: 5 -GATGATGTAGAAGCGGGAGATAC-3 .
