Gene Expression Analysis by Real-Time RT-PCR

Gene expression analysis by quantitative qRT-PCR was performed as previously described (Caruso et al., 2019b) with slight modifications. In brief, the concentration of total RNA recovered by using RNeasy Mini Kit from 10 mg of hippocampus tissue was determined by measuring the absorbance at 260 nm with a Nano Drop^® ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA). SuperScript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific) was used to carry out the reverse transcription (100 ng of total RNA for each sample), by random priming. All samples were then quantified with a NanoDrop^® ND-1000, diluted to a final concentration of 25 ng/µL, and the gene expression was simultaneously measured for all the samples by using a 384-well plates and a LightCycler^® 480 System (Roche Molecular Systems, Inc., Pleasanton, CA, USA). The QuantiTect Primer Assays (Qiagen, Hilden, Germany) employed for gene expression analysis along with official name, official symbol, alternative titles/symbols, detected transcript, amplicon length, and primers catalogue number are shown in Table 1 .

List of primers used for quantitative real-time PCR (qRT-PCR).

^# https://www.ncbi.nlm.nih.gov/gene/

^§ https://www.qiagen.com/it/shop/pcr/real-time-pcr-enzymes-and-kits/two-step-qrt-pcr/quantitect-primer-assays/

For each sample amplification, performed in quadruplicate, a total reaction volume of 10 μL, consisting of 6 μL of amplification mixture (5 μL PCR Master Mix + 1 μL specific primers) plus 4 μL of cDNA (100 ng), was used. Amplification conditions and fluorescence data collection included a first cycle at 95°C (15 min) followed by 50 cycles at 94°C (15 s), an annealing step at 56°C (30 s), and a final cycle at 72°C (30 s). As a negative control, a reaction in absence of cDNA (no template control, NTC) was performed. The relative RNA expression level for each sample was calculated using the 2^−ΔΔCT method by comparing the threshold cycle (CT) value of the gene of interest with the CT value of our selected internal control (GAPDH gene).