Quantitative Analysis of Yeast Transcripts

Total RNA was isolated from 5 × 10⁸ yeast cells of each sample using TRIzol (Tiangen Biotech, Beijing, China). Genomic DNA elimination and first-strand cDNAs generation were performed using PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time, TaKaRa Code. DRR047, Dalian, China). The RT-qPCR reactions were carried out in a total volume of 20 μL, containing 10 μL of SYBR Premix Ex Taq^TM (TaKaRa Code. RR820), 0.4 μL of each primer (10 μM), 0.4 μL of ROX Reference Dye, 2 μL of cDNA, and 6.8 μL of RNase-free water. An ABI Step One Plus Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA) was used. The primers used for amplification were designed using Primer Express software 3.0 (Applied Biosystems), and the sequences were listed in Table 3. Transcript levels were normalized against the 5.8S rRNA (GenBank accession number AB568347.1) according to Li et al. [40 (link)] and relative expression levels were calculated using the 2^−ΔΔCT method [41 (link)].