Antifungal Response in Yeast Species

S. cerevisiae strains were grown in SDglu medium at 30°C for 24 h, with or without 20 μg/ml fluconazole. C. glabrata and C. albicans cells were incubated for 8 h at 37°C or 30°C in RPMI medium, with or without 1 μg/ml fluconazole. Cells were washed with ice-cold water, resuspended in TRIzol (Thermo, Fisher), and broken using glass beads and a FastPrep machine (MP Biomedicals). RNA was extracted by respective addition of chloroform and isopropanol and washed three times with 70% ethanol. Equal amounts of RNA were treated with a DNase enzyme (New England Biolabs) and converted to cDNA (iScript cDNA synthesis kit; Bio-Rad). Real-time quantitative PCR (qPCR) reactions were conducted using GoTaq polymerase (Promega) and a StepOnePlus real-time PCR device (Thermo, Fisher). Data were analyzed using qBasePlus software (Biogazelle) (79 (link)). Further data analysis and statistics analysis of log₂(Y) transformed expression values were performed with Graphpad Prism. Transformation of the data points was performed to enable the use of standard statistical methods. Graphs show the means of the transformed values, together with their SEM. The statistical method used is mentioned under each figure. Copy number analysis of the genomic DNA of transformants was performed by qPCR, as described above.