Real-time PCR was performed using SYBR Green intercalating dye and primers. The primers were designed using the IDT PrimerQuest Primer Design Tool and synthesized by IDT. Relative gene expression (target gene expression normalized to the expression of the endogenous control gene) was calculated using the comparative Ct method (2−ΔΔCt).
The researchers used a range of IDT PrimeTime qPCR Primers and Probes for this study. In some cases, PrimeTime qPCR Primers were used with the intercalation dye, SYBR Green Dye. In other cases PrimeTime qPCR Probes were used. The probe targeting TAC-1 (SP) included the ZEN Quencher to create a ZEN-IBFQ Double-Quenched Probe for TAC-1 (SP) detection.
PrimeTime qPCR Primers and the intercalation dye, BRYT Green dye, were used to determine the initial amount of RNA present in samples. This amount was normalized using a B-actin endogenous control assay. The PrimeTime Primers utilized were designed to span the intron splice junction (thus avoiding amplification of genomic DNA) between IL-1β exon locations 2–4, BDNF exon locations 2–5, NR1 exon locations 2–3, NR2C exon locations 6–7, and β-actin exon locations 5–6. Gene expression levels were calculated from the qPCR data by normalizing the amplification rate of the target gene's expression against the amplification rate of β-actin, using the DART-PCR method, which does not assume a perfect amplification efficiency for all cycles.