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).