RNA extractions/ Reverse Transcription and Real-time quantitative PCR
Total RNA was isolated from the anterior and posterior hypothalamic area using TRIzol reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). In short, frozen pieces of tissue (~0.02 g) were homogenized in 0.5 ml TRIzol reagent in a TissueLyser II (Qiagen, Hilden, Germany) (2 x 2 min at 30 Hz) using tubes containing a 5 mm RNase free stainless-steel bead. Subsequently, 0.1 ml chloroform was added for phase separation. Following RNA precipitation by 0.25 ml of 100% isopropanol, the obtained RNA pellet was washed with 0.5 ml of 75% ethanol. Depending on size, RNA pellets were diluted in adequate volumes of RNase-free H2O (range 20-100 µL) and quantified on a Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). After DNA removal by DNase I treatment (Invitrogen, Carlsbad, CA, USA), equal quantity of RNA from each sample was used for cDNA synthesis by using RevertAid H minus first strand cDNA synthesis reagents (Thermoscientific, Waltham, MA, USA). Reverse transcription (RT; 20 µL) reactions were prepared using 1 µg RNA, 100 µM Oligo(dT)18, 5x Reaction buffer, 20 U/µL RiboLock RNase Inhibitor, 10 mM dNTP Mix, RevertAid H Minus M-MuLV Reverse Transcriptase (200 U/µL). Concentrations used for RT reactions can be found in supplementary information (Table S1). RNA was reversed transcribed by using a thermal cycler (S1000; Bio-Rad, Hercules, CA, USA). Incubation conditions use for RT were: 45°C for 60 minutes followed by 70°C for 5 minutes. Transcript levels were quantified by Real-Time qPCR using syBR Green (KAPA SYBR FAST qPCR Master Mix, Kapa Biosystems). 20 μL (2 μL cDNA + 18 μL Mastermix) reactions were carried out in duplo for each sample by using 96-well plates in a Fast Real-Time PCR System (StepOnePlus; Applied Biosystems, Waltham, MA, USA) (Table S2). Primers for genes of interest were designed using Primer-BLAST (NCBI) and optimized annealing temperature (Tm) and primer concentration. All primers were designed based on the annotatedMicrotus ochrogaster genome (NCBI:txid79684, GCA_000317375.1), and subsequently checked for gene specificity in the genomes of the common vole, Microtus arvalis (NCBI:txid47230, GCA_007455615.1), and the tundra vole, Microtus oeconomus (NCBI:txid64717, GCA_007455595.1), which both have been sequenced as a collaborative effort between the Hut-lab (Groningen), the Hazlerigg-lab (Tromsø), and the Sandve-lab (Oslo) (Table S3). Relative mRNA expression levels were calculated based on the ΔΔCT method using Gapdh as reference (housekeeping) gene (Pfaffl 2001).