Total RNA Extraction and cDNA Synthesis

Total RNA was extracted using the RNeasy plant mini kit with the automated QIAcube (Qiagen, Valencia, CA, USA). Samples were ground in liquid N₂ with a mortar and pestle. RLT buffer containing 1% beta-mercaptoethanol and 1% polyvinylpyrrolidone was added to 50 mL tubes containing ground tissue and vortexed thoroughly. Following suspension in the modified RLT buffer, 0.4 volumes 5 M potassium acetate, pH 6.5 was added to the buffer, mixed by inverting and incubated for 15 min on ice. Samples were centrifuged for 15 min at 15,000 g at 4°C. Supernatant was then loaded into the QIAcube and RNA extraction was performed with an on-column DNAse I (Qiagen, Valencia, CA, USA) digestion. RNA quality and quantity was assessed with microfluidics using the Experion™ automated electrophoresis system and RNA StdSens chips (Bio-Rad, Hercules, CA, USA). cDNA synthesis reactions were performed with 1 μg of total RNA and oligodT primers according to manufacturer’s instructions (RevertAid, Fermentas Inc., Glen Burnie, MD, USA). Separate reactions were performed for 18S rRNA using random primers instead of oligodT primers. The cDNA from triplicate first strand cDNA reactions was pooled and served as the template for triplicate technical qPCR reactions with the Maxima SYBR green qPCR master mix (Fermentas Inc., Glen Burnie, MD, USA) and detected with the iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Cycling conditions consisted of 10 min at 95°C followed by 40 cycles of 15 sec at 95°C and 1 min at the optimum annealing temperature (Table2).