Step one plus kit

Real-time PCR was performed using a Step One Plus kit (Applied Biosystems, Foster City, CA, USA). Primers and TaqMan probes specific for the target genes (TaqMan Gene Expression Assay) were obtained from Applied Biosystems (Foster City, CA, USA). The expression levels of target neurotrophins (BDNF and NT-4/5), the neurotrophin receptor (TrkB), and a marker of neuronal activity (c-fos) were assayed using β-actin as an internal control. Individual codes of TaqMan Gene Expression Assays were as follows: BDNF (Mm334042), NT-4 (Mm01701591), TrkB (Mm00435422), c-fos (Mm01302932), and β-actin (4352341E). Individual reactions contained 0.5 µL of target primer, 0.5 µL of β-actin primer (TaqMan Gene Expression Assay, Applied Biosystems), 3 µL of RNase/DNase-free H2O, 5 µL of TaqMan Gene Expression Master Mix (Applied Biosystems), and 1 µL of sample cDNA in a total volume of 10 µL. The thermal profile was a single cycle of 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s.
The relative expression level of each target gene was determined relative to the transcript of β-actin using the comparative (ΔΔCt) method. Averages of triplicate Ct values were used for the analysis. Transcript abundance was normalized to the average of the adult control group for each target gene.