Fastprep r 24 cell disrupter

Fungal mycelia were homogenized in 1 ml of peqGOLDTriFast DNA/RNA/protein purification system reagent (PEQLAB Biotechnologie, Erlangen, Germany) using a FastPrep(R)-24 cell disrupter (MP Biomedicals, Santa Ana, CA, USA). RNA was isolated according to the manufacturer’s instructions, and the concentration was measured using the NanoDrop 1000 (Thermo Scientific, Waltham, US). Synthesis of cDNA from mRNA was carried out using the RevertAid^TM H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, USA) according to the manufacturer’s instructions. Quantitative, reverse transcription PCRs (RT-qPCRs) were performed in a Rotor-Gene Q system (Qiagen, Hilden, Germany). All reactions were performed in triplicates. The reaction mixture (final volume 15 µl) contained 7.5 µl 2 x iQ SYBR Green Mix (Bio-Rad, Hercules, USA), 100 nM forward and reverse primer and 2.5 µl cDNA (diluted 1:20). Primer sequences are provided in (Table 1). Cycling conditions and control reactions were performed as described previously [24 (link)]. Data normalization using sar1 and act as reference genes and calculations were performed as published previously [24 (link)].