The CFX 96-c1000 Thermocycler (Bio- Rad Laboratories, Mississauga, ON, Canada) was used to quantify INF-γ mRNA expression. The INF-γ gene expression was quantified relative to the mRNA expression of the housekeeping gene, β-actin. The qPCR assays for both the β- actin and INF-γ genes were run on the same plate. Fast SYBR® Green Master Mix (Invitrogen, Burlington, ON, Canada) was used. The primers for the INF-γ gene (F-ACACTGACAAGTCAAAGCCGCACA, R-AGTCGTTCATCGGGACCTTGGC) [39 (link)] and primers for the β-actin gene (F-CAACACAGTGCTGTCTGGTGGTA, R-ATCGTACTCCTGCTTGCTGATCC) [39 (link)] were used in each reaction. Each reaction volume consisted of 10 µL of SYBR Green master mix, 200 ng of cDNA of respective samples as a template, and 0.5 µL of forward and reverse specific primers that targeted the genes. The qPCR conditions were 95 °C for 20 s of pre-incubation and 95 °C for 3 s. For 40 amplification cycles, it was 60 °C for 30 s. Then, 95 °C and 65 °C with a 0.5 °C rise in temperature every 5 s was used for the melting curve analysis.
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