Quantitative RNA Extraction and RT-qPCR Analysis

Total RNA was extracted from yeast cells using a total RNA Mini Kit (Geneaid) as described [56 (link), 57 (link)]. To remove potential contamination of genomic DNA, total purified RNA was incubated with RQ1 (Promega) DNase at 37°C for 40 min and then inactivated at 65°C for 10 min. Reverse transcription involved a cDNA synthesis kit (Epicentre) in an RNase-free environment. RNA underwent initial incubation at 65°C for 5 min with oligo-dT. Then, the reaction mixture including buffer, dNTPs, DTT, ribonuclease inhibitor, and MMLV reverse transcriptase was incubated at 37°C for 60 min and then inactivated at 85°C for 5 min. cDNA prepared from triplicate biological samples was used for PCR analysis. Real-time quantitative PCR (qPCR) involved use of Stratagene Mx3000p (Agilent Technologies) and Kapa SYBR Fast Universal qPCR Kit (Kapa Biosystems). The primers used in this study are available upon request.