Evagreen mix

Transcript levels were analyzed by qPCR, for which 1 μg of total RNA was treated with DNase I (Thermo Fischer Scientific, Waltham, MA, USA) to eliminate gDNA contamination. The Superscript II RT system (Invitrogen, Carlsbad, CA, USA) was used for complementary DNA synthesis, according to the manufacturer’s instructions. Levels of transcripts were quantified for six selected DEGs of interest using peach fruit flesh of normal and slow ripening individuals. Every reaction was performed on an Eco system (Illumina Inc.) with Evagreen mix (Biotium, Fremont, CA, USA) and specific primers.
Three biological replicates and three technical replicates were used for each gene, and RPII was used as a control [59 (link)]. The PCR program was (i) enzyme activation at 95 °C for 10 min, with 40 cycles of (ii) 95 °C for 15 s, annealing for 15 s, and 72 °C for 15 s. After every PCR, a melting curve was generated from 55 to 95 °C. Finally, the data were analyzed with GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA), and standard error was used for the biological and technical replicates. To determine the correlation between RNAseq and qPCR expression results, a Pearson correlation coefficient was calculated for each gene analyzed.