Quantitative Analysis of Gene Expression

The cDNA was synthesized from DNase-treated RNA (1,000 ng) using 0.5 μg oligo-dT_12-18, 100 U Superscript III reverse transcriptase, and 20 U RNaseOUT recombinant RNase inhibitor (all from Thermo Fischer Scientific) in a total volume of 20 μl for 2 h at 42°C followed by heat inactivation for 15 min at 70°C. Quantitative PCR with EvaGreen QPCR mix II (Bio&SELL) was performed with 1:10 diluted cDNA. Primers were used at a final concentration of 500 nM. Target gene expression was calculated using the ΔΔCT method (79 (link)), with normalization to the housekeeping genes CgACT1 for C. glabrata or ScACT1 for S. cerevisiae. For mtDNA quantification, yeast DNA was extracted following the Harju et al. protocol (80 (link)), and 100 ng was the reaction concentration. ScCOX3 and CgCOX3 were used as mitochondrial target genes and CgACT1 or ScACT1 as housekeeping genes. All experiments were done in independent biological triplicates on different days and are shown as the mean with standard deviation (SD) for each time point.