Real time qPCR
To assess and compare the expression of aquaporins across various
cultivars and treatments, RT-qPCR was conducted using 2 μL of 1:10
diluted cDNA samples in an 8 μL reaction mixture containing 600 nM
gene-group primers (Table 1 ) and 5 μL of SYBR Green Master Mix
2X (Applied Biosystems) in an Applied Biosystems 7500 Real-Time PCR
system. Amplification was performed in a two-step process, consisting of
denaturation at 95°C for 10 min, followed by 40 cycles of 15 s of
denaturation at 95°C and 1 min of annealing and extension at 60°C. A
dissociation stage was then carried out. Data collection was performed
at the end of each cycle’s step 2. These conditions were used for both
target and reference genes. The reactions were conducted in triplicate
for each sample (technical replicates) on a 96-well plate, and four
independent samples were tested for each treatment (biological
replicates). Transcript levels were calculated using the 2–ΔΔCt method
(Livak and Schmittgen, 2001) for both target and reference genes.