The QuantiFast SYBR® Green PCR Kit (Qiagen Corp., CA, USA) was used on a Rotor-Gene Q MDx 5 plex platform (QIAGEN Inc., CA, USA). Briefly, the 20 μL reaction mix was prepared by adding 10 μL of master mix, 1 μL forward primer (10 pmol), 1 μL reverse primer (10 pmol), 1 μL cDNA from the sample, and 7 μL of nuclease-free water. The PCR cycles employed the following parameters: 95°C for 5 min; 40 cycles of 95°C for 10 s followed by 30 s annealing (Table-1 for details about annealing temperature); and 72°C for 10 s with final melting at 95°C for 20 s. The detection of fluorescence emission occurred during the extension step. The β-actin gene was used as an internal control to which the fold changes in gene expression were normalized. The single target amplification specificity was approved by the melting curve. The relative quantitation was calculated using 2−DDCt analysis. Table-1 shows primer sequences that were used in the real-time RT-qPCR analysis.

Primer sequences that are used in the real-time qPCR analysis

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