Quantification of Gene Expression by qRT-PCR

Total RNA was extracted from log phase cultures grown in YEPD at 30 °C under constant agitation as described [7 (link)]. Gene expression levels were determined by real-time quantitative reverse transcription-PCR (qRT-PCR) in a StepOne real-time PCR system (Applied Biosystems, Foster City, CA, USA). cDNA was prepared with a PrimeScript RT reagent kit (Perfect Real Time) (Takara). Subsequent qPCRs were performed with a 0.2 µM concentration of each primer and a 0.1 µM concentration of TaqMan probes (see Table S1) and iTaq Supermix with ROX (Amine-reactive carboxy-x-rhodamine) (Bio-Rad, Reinach, Switzerland) according to the manufacturer’s instructions. Assays were performed in biological triplicates and normalized to ACT1 (CLUG_03241).