*2.5. qPCR Reaction*

qPCR was performed in a CFX96 Real-Time PCR Detection System (Bio-Rad). The reaction mixture (10 μL) contained 5 μL of iTaq universal SYBR Green Supermix, 0.5 μL of each primer (10 μM), 1 μL of cDNA, and 3 μL of nuclease-free water. Amplifications were performed using the following cycling profile: an initial activation step (95 ◦C for 3 min.) followed by 40 cycles of denaturation at 95 ◦C for 10 s, annealing at a temperature appropriate for selected starters (Table 2) for 10 s, and extension at 72 ◦C for 20 s. For melting curve analysis, a dissociation step cycle (60 ◦C for 5 s, and then 0.5 ◦C for 5 s until 95 ◦C) was added. All of the qRT-PCR experiments were performed in three technical replicas.


**Table 2.** Starters and temperatures of annealing selected for expression analysis.

Analysis of gene expression was done through a comparative method (ΔΔCt) in order to determine the relative level of expression of selected mRNAs. This method is based on calculating the di fferences in the level of expression of the test gene and the reference gene. The calculations use the threshold cycle (Ct) values of the qPCR reaction. Ct values were determined for both the test and reference genes in both the test and control samples, for which the di fferences between the individual Ct values ( ΔCt) were then calculated.
