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.