Rest 2009

Tissue samples were frozen in liquid nitrogen and ground in a mortar. Total RNA was isolated using the Direct-zol RNA MiniPrep Plus kit with TRI Reagent (Zymo Research, Irvine, CA, USA) and followed by DNase I treatment (Thermo Fisher Scientific). Lack of DNA contamination was verified by PCR and qPCR on the RT control. cDNA was synthesized from 1 µg of RNA using the iScript cDNA Synthesis Kit (Bio-Rad). Real-time quantitative PCR (qPCR) was conducted using the StepOnePlus (Applied Biosystems) thermocycler with the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific). Primers were validated for single product specificity and their effectiveness to range between 90 and 105% (Electronic Suppl. Table S1). qPCR thermal cycling conditions were: initial denaturation at 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 55 °C for 60 s. Amplification was followed by melt curve analysis to verify single product amplification. Normalization was done to the expression of the actin gene. qPCR reactions were done in at least three biological replications. Relative gene expression was calculated using the REST 2009 (Qiagen) software.

Progenitor cells were cultured with and without hanging inserts in 12-well plates for 7 and 21 DIV before RNA extraction using the RNeasy mini kit (Qiagen, 74106). Following cDNA synthesis from the RNA samples, qPCR reactions were set up using TaqMan gene expression assays for Tuj1 (Hs00801390_s1), KCC2 (Hs00221168_m1) and GABA A1 receptor subunit (Hs00971228_m1) using the CFX Connect™ Real-Time PCR Detection System (BioRad). Experiments were set up in triplicates. qPCR data from three experimental repeats were analyzed using REST 2009 (Qiagen) with Tuj1 as the reference gene. All values were normalized to the ‘no insert’ values for analysis.

For qRT-PCR analysis, cDNA was synthesized using SuperScript (Invitrogen, Carlsbad, CA, USA) with oligo(dT)s, using 500 ng of total RNA for each sample. Three biological replicates and three technical replicates were used for each time point validation (nine replicates in total). Primers were designed based on SAS sequences using the Primer Express 2.0 (Applied Biosystems, Foster City, CA, USA) software, and only primers with an amplification efficiency between 90 and 110% were used (Supplementary Table S14). Reaction mixtures consisted of 5 μL of 1.5 μM primer mix, 1 μL of cDNA (diluted 1:10) and 6 μL of Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Reactions were performed on 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). For primer SCSGLR1045D05.g, primer concentration was reduced to 312.5 nM. Expression ratio was estimated using REST 2009 (Relative Expression Software Tool, Qiagen, Hilden, Germany) as described in [107 (link)]. The endogenous genes for each tissue were identified using the geNorm software [108 (link)] (Supplementary Table S15).

Total RNA was extracted from cells with a Trizol reagent (Invitrogen, Thermo Fisher, Waltham, MA, United States) according to the manufacturer’s instructions [46 (link)]. RNA yield and purity were checked using a Nanodrop spectrophotometer (EuroClone, Milan, Italy), and total RNA from each sample was reverse transcribed into cDNA using Moloney Murine Leukemia Virus Reverse Transcriptase (Sigma-Aldrich). Real-time PCR was performed on a StepOne™ Real-Time PCR System (Thermo Fisher, Waltham, MA, USA) using SensiFAST SYBR Hi-Rox (Bioline, LABGENE SCIENTIFIC SAZI, Châtel-Saint-Denis, Switzerland). The comparative Ct method (ΔΔCt) was used to quantify gene expression, and the relative quantification was calculated as 2−ΔΔCt. The presence of non-specific amplification products was excluded by melting curve analysis. Statistical analyses on real-time PCR data were performed using the Relative Expression Software Tool (REST2009, Qiagen, Venlo, The Netherlands) [47 (link)]. The forward and reverse primer sequences are shown in Table 1.

4.1. Extracting RNA and cDNA synthesize
High pure RNA tissue kits were employed to extract total RNA as per the manufacturer's protocol (Roche, Switzerland). The reverse transcriptase of Moloney murine leukemia virus (Fermentas, Lithuania) was then used in the presence of RNase inhibitors and random hexamers for the transcription of 1 μg of total RNA into cDNA.
4.2. Quantitative Real-Time PCR According to Table-1, specific primers for MAP1ALC3, BECN1, CASP8, BAX, and TP53 genes were employed to perform quantitative real-time PCR considering GAPDH the internal control. Reactions were conducted by utilizing SYBR® Premix Ex Taq II (Takara, Japan) and a RotorGene™ 6000 machine (Qiagen, Germany).Moreover, initial denaturation was carried out for 15 minutes at 95°C followed by 40 denaturation cycles at the same temperature for five seconds in primer-specific conditions and extension for 20 seconds at 60°C. The candidate groups were compared in terms of the results of quantitative PCR in REST-2009 (Qiagen, Germany).