Quantitative RT-PCR Analysis of Gene Expression

Tissue procurement has already been described [41 (link)]. Briefly, for the extraction of RNA, one snap-frozen cerebrum hemisphere per animal was homogenized in peqGOLD RNAPure™ (PEQLAB Biotechnologie, Erlangen, Germany), and the total RNA was isolated per the product protocol. Then 2 µg of RNA was DNase treated and reverse transcribed. Each cDNA sample was diluted 10 times with nuclease-free water and was stored at −20 °C.
PCR was performed with qPCR BIO Mix Hi-ROX (NIPPON Genetics Europe, Düren, Germany). The amplification program was as follows: 50° for 2 min, 94 °C for 2 min, 40 cycles at 94 °C for 5 s, and 62 °C for 25 s. Reactions for each sample were carried out in triplicate in 96-well plates. The detection of PCR products was performed in triplicate in 11 μL reaction mix, each containing 5 μL of qPCR mastermix, 2.5 μL of 1.25 μM of each oligonucleotide primer, 0.5 μL of 5 μM of probe, and 3 μL of cDNA template (17 ng). Probes were labeled with the fluorescent reporter 6-carboxy-fluorescein (6-FAM) at the 5′ end and the fluorescent quencher carboxytetramethylrhodamine (TAMRA) at the 3′ end (BioTez Berlin Buch GmbH, Berlin, Germany). The PCR products of target genes were quantified in real time with the sequences summarized in Table 1. The abundance of each gene was determined relative to the hypoxanthine-guanine phosphoribosyl-transferase (HPRT). The expression of target genes was analyzed with the StepOnePlus real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) according to the 2^−ΔΔCT method [45 (link)].