Oligo dt 23 primers

The ethanol/phenol fixed cells were resuspended in 1 mL TRI reagent (Sigma-Aldrich, Darmstadt, Germany), lysed using glass beads as described in the previous section and RNA was extracted according to the TRI reagent protocol. DNase treatment was carried out using the Ambion DNA-free kit (Thermo Fisher Scientific, Vienna, Austria). RNA concentration and integrity were analyzed using the Nanodrop spectrophotometer and Bioanalyzer (Agilent, Vienna, Austria). cDNA was synthesized using Oligo(dt)₂₃ primers (NEB, Frankfurt, Germany) and Biozym cDNA synthesis kit (Biozym, Vienna, Austria). Quantitative PCR was carried out using Biozym Blue Probe qPCR kit on a Rotor Gene Q instrument (Qiagen, Hilden, Germany). Changes in transcript levels were calculated relative to the reference sample, after normalization to ACT1 (PP7435_Chr3-0993) expression, using the threshold cycle method of the Rotor Gene software.

For transcript level analysis, three clones per strain were cultivated in YPD medium (yeast extract 1%, peptone 2%, glucose 2%) in shake flasks overnight. Cells were washed and used to inoculate YNB medium (YNB without amino acids and ammonium sulfate 3.4 g L⁻¹, ammonium sulfate 10 g L⁻¹, potassium phosphate buffer 0.1 mol L⁻¹ pH 6, biotin 0.4 mg L⁻¹) with 2% glucose or xylose as carbon source. After 6 h of cultivation at 25 °C, cells were harvested and RNA was extracted according to the TRI reagent (Sigma-Aldrich) protocol, followed by DNase treatment with the Ambion DNA-free kit (Invitrogen) and cDNA synthesis with oligo(dT)23 primers (NEB) and the Biozym cDNA synthesis kit. RNA integrity was analyzed by agarose gel electrophoresis. Quantitative PCR was performed on a Rotor-Gene Q instrument (Qiagen) using the Blue S´Green qPCR kit (Biozym). Transcript levels were normalized to ACT1 expression and relative expression levels were calculated using the average expression of SOR1 in K. phaffii X-33 grown in YNB glucose as reference.