Quantitative real-time polymerase chain reaction (RT-PCR).
Quantitative real-time RT-PCR for IFN- γ gene was performed on a Stratagene Mx3005P qPCR System (Agilent Technologies, Germany) using the qPCR Green Master (Jena Bioscience, Germany). PCR reaction with 20 μl final volume was prepared by adding 10 μl qPCR Green Master, 0.5 μl forward primer (10 μM), 0.5 μl reverse primer (10 μM) (Table I), 5 μl template cDNA and 4 μl PCR grade water into real time PCR wells. The cycling program was prepared as following 95º C for 10 min, then 40 cycles (95º C for 15 secs, 58º C for 1 min). The transcription levels of target genes were normalized to those of the β-actin gene, which used as the reference gene. The normalized quantity of the target gene was obtained by subtracting the cycle threshold (CT) for β -actin from the CT for the target gene (ΔCT sample). The same calculation was performed with controls (ΔCT control). Then ΔΔCT was calculated as the difference of these values (ΔΔCT = ΔCT sample – ΔCT control). Finally, the relative expression was expressed as fold change by a 2−ΔΔCT relative to control.
Table I: Primer used for Quantitative real-time RT-PCR for IFN- γ gene.