RNA Extraction, cDNA Synthesis and Reverse Transcriptase-Quantitative PCR (RT-qPCR)

For all experiments, 100 mg leaf tissue was harvested from each of the nine treatments (3 water stress × 3 aphid infestations), flash frozen, and stored at -80°C for further processing. Plant RNA was extracted using the Trizol^® (Invitrogen, Grand Island, NY, USA) method, checked for purity and quantity using a Nanodrop ND 100 (Thermo Scientific, Pittsburgh, PA, USA). RNA was then treated with Turbo DNase^® (Invitrogen, Grand Island, NY, USA) in order to remove DNA contamination. Complete removal of DNA was verified by PCR using DNase treated RNA as template for amplification with the internal control FBOX gene (Le et al., 2012). Two micrograms of RNA was used as a template for cDNA synthesis using the Verso^® cDNA synthesis kit (Thermo Scientific, Pittsburgh, PA, USA).

RT-qPCR was performed using SYBR Green^® (Biorad, Berkeley, CA, USA) on a CFX Connect^® (Biorad, Berkeley, CA, USA) thermocycler. The cycling conditions used were: 95°C for 2 min, followed by 40 cycles of 95°C for 30 s, and 60°C for 30 s. PCR efficiencies (E) of target and internal control genes were determined using the LinRegPCR software (Ruijter et al., 2009) and are shown in Table ​Table11. Reactions for all samples were performed in duplicate and three biological replicates and a negative and positive control were used in each run. Fold change was determined by normalizing transcript levels of the genes of interest to the internal control gene (FBOX), followed by normalization to expression of the respective gene in a plant that was not subjected to water stress or aphid infestation using the following formula, 2^-ΔΔCT (Livak and Schmittgen, 2001). Fold changes were log₂ transformed in order to normalize data. Log₂ (fold change) data is presented and also used for all statistical analysis.