Quantifying Antifungal Drug Resistance in Candida using qRT-PCR

The qRT-PCR analysis was performed as described previously (21 (link)), with minor modifications. Isolates were incubated without any drug or with PG alone, FLC alone, or PG+FLC at synergistic concentrations at 37°C overnight. The suspensions were adjusted to 5 × 10⁷ cells/ml in PBS, and the supernatants were collected after centrifugation at 3,000 × g. Total RNA was isolated using a yeast RNAiso reagent kit (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. RT-PCR was performed using RevertAid first-strand cDNA synthesis kits (Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCRs for CgCDR1, CgCDR2, and CgPDR1 were run in triplicate using SYBR green real-time PCR master mix kits (Toyobo, Osaka, Japan) in an ABI 7500 real-time fluorescent quantitative PCR system (Applied Biosystems, Foster City, CA, USA). The primers used in this study are listed in Table 4. Each qRT-PCR mixture (25 μl) contained 12.5 μl SYBR green real-time PCR master mix, 9.5 μl double-distilled water, 2 μl each primer, and 1 μl cDNA. PCR conditions were as follows: initial denaturation at 95°C for 1 min, followed by 40 cycles of 15 s at 95°C, 15 s at 60°C, and 45 s at 72°C. Target gene expression was quantified using the 2^–ΔΔCT method, with ACT1 as a control (39 (link)).