*2.7. Expression Analysis of Different Genes*

To examine the temporal expression patterns of selected genes, qRT-PCR was performed. The qRT-PCR was performed in a CFX-96 Real-time PCR Detection 4 System (Bio-Rad, Hercules, CA, USA). Reactions were conducted in a total volume of 20 pl using 50 ng of cDNA, 10 pmol of forward and reverse primers, and 10 L of 2× Sso Fast Eva GreenqPCR Supermix (Bio-Rad, Hercules, CA, USA). The cycling conditions were as per the manufacturer's protocol with a primer-specific annealing temperature. The threshold cycle (Ct) was automatically determined for each reaction using the system set with default parameters. The transcript levels were normalized to the actin transcript, and the fold differences of each amplified product in the samples were calculated using the 2-AACt method.
