Specific primer pairs (Table 1) for the 18 selected candidate reference genes (and also for the target genes AGPL and AGPS) were designed using Oligo v7.60 software (Molecular Biology Insights). To ensure maximum specificity and efficiency in PCR amplification, highly stringent conditions were initially selected for primer design. For each primer pair these were: ΔG of the most stable 3'-dimer > -3.5 Kcal/mol, ΔG of the most stable dimer overall > -7.5 Kcal/mol, no hairpins with ΔG < -1 Kcal/mol and a melting temperature (Tm) > 40°C, primer efficiency (PE) > 480, PE of false priming sites < 100, optimal annealing temperature (Ta) of the PCR product between 62–65°C, primer lengths of 20–28 nucleotides, and amplicon length ranging between 100–200 base pairs. When most of the criteria were successfully met, the recommended temperature for annealing/extension in PCR was 68°C. All designed primers were finally ordered from a commercial supplier (Eurofins). Appropriate performance of each primer pair was initially tested by PCR amplification of the target amplicons employing the same conditions described below for RT-qPCR. Specificity of each primer pair was verified by melting curve analysis from 70°C to 95°C with a ramp speed of 0.5°C every 10 s. Single, sharp peaks were obtained in all instances, thus ruling out amplification of non-specific products or primer dimer artifacts (S3 Fig). To determine real-time PCR efficiency (E) of each primer pair, standard curves were prepared from serial dilutions of cDNA (from 100 ng to 0.01 ng) and the results plotted in Excel against their CT values (S4 Fig). Then, slopes of the linear regressions were determined applying the equation E = 10−1/slope as previously reported [61]. The linear correlation coefficient (R2) of each primer pair was also determined in Excel. The PCR products were analyzed by standard agarose gel electrophoresis (2.5% w/v in TAE 1X) and clear DNA bands of the expected sizes were obtained (S5 Fig).

a Ta represents annealing temperature.

RT-qPCR was conducted using a CFX96™ Real-Time PCR Detection System (Bio-Rad). Each 10 μL reaction contained 5 μL of 2X iQ™ SYBR® Green Supermix (Bio-Rad), 300 nM of both forward and reverse primers (0.3 μL of a 10 μM stock each), 2 μL of cDNA (corresponding to the cDNA retrotranscribed from 10 ng of RNA), and 2.4 μL of nuclease-free water. All reactions were run in duplicate, and the mean threshold cycle (CT) of each sample (S3 Table) was used for further calculations. Relative transcript levels of the target genes were determined using the 2-ΔΔCt method [62]. The thermal cycling profile included an initial incubation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 68°C for 30 s.

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