Evagreen 5 qpcr rox mix

Mycelia from specified cultivations were briefly washed with water and ground under liquid nitrogen. RNA was isolated using the RiboPure RNA Purification Kit (Ambion, Life Technologies, Darmstadt, Germany). Residual genomic DNA (gDNA) was removed by the DNA-free kit (Ambion). cDNA was synthesised by Revert Aid Reverse transcriptase (Thermo Scientific, Schwerte, Germany) using anchored oligodT primers. qRT-PCR was carried out on a CFX384 Touch Real-Time PCR Detection System (BioRad, Munich, Germany) using the EvaGreen 5 × QPCR (ROX) Mix (Bio & Sell, Feucht, Germany) following the manufacturer's protocol and using 1:5 and 1:10 dilutions of cDNA samples serving as templates. The actin gene (actA, ATEG_06973) and the enolase gene (enoA, ATEG_02902) were used for normalisation of transcript levels yielding similar results. Normalised transcript levels were calculated as fold expression = 2^{Δ(reference − target)}. Primers used for qRT-PCRs showed a primer efficiency of 1.89–2.0 and are listed in Figure 4—source data 2 (Figure 4—source data 2).

C. albicans yeast cells were incubated for 4 or 20 h at 37°C in MOPS-buffered RPMI 2% glucose medium in presence or absence of 10 mg/ml TC. Cells were harvested by centrifugation, directly frozen in liquid nitrogen and RNA was extracted as described above. cDNA was produced by RevertAid Reverse Transcriptase (Thermo Scientific) with anchored oligo(dT) primers. qRT-PCR was analyzed on a CFX384 Touch Real-Time PCR Detection System (BioRad, Munich, Germany) using EvaGreen 5 × qPCR (ROX) mix (Bio & Sell, Feucht, Germany). The ACT1 and EFB1 genes served as reference for normalization. Normalized transcript levels were expressed as fold expression = Δ2^{(reference-target)} based on expression levels in absence of TC. Oligonucleotide sequences are provided in Table 1.