*4.6. cDNA Synthesis and qRT-PCR*

According to the manufacturer's instructions, total RNA was retro-transcripted employing gene-specific primer and random primers of Invitrogen™ SuperScript™ II Reverse Transcriptase (Thermo Fisher Scientific). cDNA was used as a template for qRT-PCR reactions. The PCR reactions were performed in technical triplicates with the LightCycler 480 SYBR Green I Master reagent using the LightCycler® 480 Instrument (Roche, Italy) in 96-well reaction plates. PCR conditions were: one cycle at 95 ◦C for 5 min, followed by 40 cycles of 95 ◦C for 15 s and 60 ◦C for 30 s. At the end of the PCR, to confirm the presence of a unique amplicon, the melting curve was evaluated and a single peak in every reaction was observed. Relative template abundance was quantified using the standard curve method [73] and the *Elongation Factor 1-Alpha* (Accession: AY338249.1) was used as a reference gene for expression normalization. PCR efficiency was estimated using six-point, 10-fold, diluted standard curves. Means from two independent replicates were subjected to SD calculation and Student's t-test. The primers were designed using the Primer3 software web version 4.1.0 (Table 1).

**Table 1.** The sequences of primers of PRs pear genes and erw genes, used in the qRT-PCR reactions.

