Total RNA isolation and cDNA synthesis

Total RNA was isolated and quantified as described in Goulao et al. (2012) for each of the following 24 conditions: three genotypes (CL153, Icatu, IPR108), four temperature regimes (25/20°C, 31/25°C, 37/30°C, 42/34°C), and two [CO₂] (380, 700 μL CO₂ L⁻¹), using the RNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. In order to avoid eventual genomic DNA contaminations, RNA samples were digested with Ambion® DNA-free™ DNase (Ambion, USA). RNA integrity was verified in 1.5% agarose—TAE gel electrophoresis containing GelRed Nucleic Acid Gel Stain (Biotium, USA). All RNA samples were individually analyzed for the possible presence of DNA contamination by standard PCR reactions (35 cycles) using primers designed for ubiquitin (UBQ) gene (Table ​(Table1),1), in the absence of cDNA synthesis. Total RNA concentration and purity were further verified through BioDrop Cuvette (BioDrop, UK) measurements to guarantee the same amount of starting material in subsequent cDNA synthesis. RNA concentrations ranged between 333 and 1,056 ng/μl, with OD 260:280 ratios always above 1.98.

Primer sequences and amplicon characteristics for each of the 10 candidate reference genes under evaluation.

One microgram of DNA-free total RNA was used to synthesize first-strand cDNAs using oligo-(dT)₁₈ primer and the SuperScript II first-strand synthesis system (Invitrogen, USA).