RNA extraction, cDNA synthesis, and quantitative real-time reverse transcription polymerase chain reaction

Dorsal hippocampi were dissected and immediately stored at −80°C in RNAlater solution (Ambion) for later isolation of total RNA. For RNA extraction, Qiazol (Qiagen) was added to the hippocampal tissues and they were homogenized using stainless steel beads (Qiagen). Chloroform was then added to the homogenates, and the samples were centrifuged at 12,000g at room temperature for 15 min. RNA was precipitated from the aqueous phase using ethanol and then cleaned using an RNeasy kit (Qiagen). RNA was eluted in nuclease-free water and treated with DNase (Qiagen) at room temperature for 25 min. The cleaned RNA was precipitated in ethanol, sodium acetate (pH 5.2), and glycogen overnight at −20°C. RNA samples were centrifuged at top speed at room temperature for 20 min. The precipitates were further washed with 70% ethanol and centrifuged at top speed for 5 min. The RNA precipitates were dried and resuspended in nuclease-free water, and concentrations were estimated using a NanoDrop (Thermo Fisher Scientific). Complementary DNAs (cDNAs) were prepared from 1 μg of RNA using the SuperScript IV First-Strand Synthesis System (Ambion). Real-time reverse transcription polymerase chain reactions (RT-PCRs) were performed in a 384-well optical reaction plate with optical adhesive covers (Life Technologies). Each reaction was composed of 2.25 μl of cDNA (2 ng/μl), 2.5 μl of Fast SYBR Green Master Mix (Thermo Fisher Scientific), and 0.25 μl of primer mix (IDT). A minimum of three technical replicates per reaction was performed on the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, Life Technologies). Data were normalized to housekeeping genes (Tubulin, Pgk1, and B2m), and 2^(−ΔΔCt) method was used for gene expression analysis.