Quantitative RT-PCR analysis of gene expression in yeast

Total RNA was isolated from yeast cells using TRIzol (Invitrogen), and further purified with the kit RNeasy (Qiagen), following the manufacture's instruction. The cDNA was synthesized using the SuperScript system (Invitrogen). Following synthesis, cDNA purification was conducted after RNA degradation using adsorption chromatography, as described (Trujillo-Esquivel et al., 2016 (link)). Absence of genomic DNA in the cDNA preparations was confirmed by amplification of the ACT1 gene which contains an intron of 658 bp (data not shown).
All primer pairs used in qPCR reactions are listed in Table S4. The reaction mixtures were prepared using the SYBR® Green PCR Master Mix (Thermo Fisher Scientific) and analyzed in a StepOnePlus Real-Time PCR System (Applied Biosystems). All reactions produced a single amplicon, with a uniform melting curve, as determined by the dissociation profile of the products. Relative quantification was determined by calculating 2^−ΔΔCT (Livak and Schmittgen, 2001 (link)). Expression data were normalized using RPP2B, a housekeeping gene previously used as a control in expression assays (Nailis et al., 2006 (link)).