Validating Hippocampal Gene Expression Differences in Tame and Aggressive Rats

To selectively and independently verify the tame-versus-aggressive rat hippocampal DEGs found in the present study (Table 2), we performed a qPCR control assay on the total RNA extracted only from the remaining samples of the hypothalamus of tame (n = 8) and aggressive (n = 8) rats. First, with the help of TRIzol™, we isolated total RNA, purified it on Agencourt RNAClean XP Kit magnetic beads (Beckman, #A63987), and quantified it by means of a Qubit™ 2.0 fluorometer (Invitrogen/Life Technologies), along with a high-sensitivity RNA kit (Invitrogen, cat. #Q32852). Then, we synthesized cDNA using a reverse transcription kit (Syntol, #OT-1). Next, using the PrimerBLAST web service [89 (link)], we designed oligonucleotide primers for qPCR (Table 3). Then, we carried out qPCR on a LightCycler^® 96 (Roche, Basel, Basel-Stadt, Switzerland) with an EVA Green I kit in three technical replicates. We determined qPCR efficiency by means of serial cDNA dilutions (standards). In line with the commonly accepted recommendations [90 (link)], we simultaneously analyzed two reference genes, namely Ppia (peptidylprolyl isomerase A) [91 (link)] and Rpl30 (ribosomal protein L30) [92 (link)].