Sv rna isolation

Total RNA from postmortem brain tissues was isolated using a commercially available kit (SV RNA Isolation, Promega, Madison, WI, USA). qRT-PCR was performed on a MasterCycler (Eppendorf) using a SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen, Carlsbad, CA, USA). A typical reaction mixture of a total volume of 25 μL consisted of 0.5 μL Superscript III RT/Platinum Taq mix, 12.5 μL 2X SYBR Green Reaction Mix (includes 0.4 mM of each dNTP and 6 mM MgSO₄), 12.5 pMol of each of forward or reverse primers, and 4 μL DEPC-treated water. PCR amplification was done with an initial incubation at 55°C for 1,200 sec, then at 95°C for 120 sec followed by 35 cycles of 95°C for 15 sec, 50°C for 30 sec, 72°C for 30 sec, and a final melting curve from 55°C to 95°C at 0.2°C/sec. We confirmed the primer specificity by melting curve analysis and electrophoresis of PCR products on a 2% agarose gel to confirm the presence of a single band of the predicted size. The mRNA for genes of interest was normalized to two control genes (GAPDH and β-actin) and a geometric mean of these genes. The mRNA expression levels were quantified by the delta-delta Ct method. Primers were synthesized by Integrated DNA Technologies (Additional file 1: Table S1).

Total RNA from the PFC (n = 3–7) samples was isolated using a commercially available kit (SV RNA Isolation, Promega, Madison, WI, USA). All RNA samples were quantified using a Nanodrop. After the quantification, cDNA was prepared using iScript™ cDNA Synthesis Kit (Bio-Rad CA, USA). q-RT-PCR was performed on a Master Cycler (Quant Studio-7 Real-Time PCR Systems, Thermo Fisher Scientific, USA) using iTaqTM Universal SYBR^® Green Super mix (Bio-Rad, CA, USA). Primers were synthesized by Integrated DNA Technologies. Ct values of genes of interest were normalized to that of housekeeping gene, beta2-microglobulin (B2M). The list of primers used is given in Table S1.