Confirmation of Data Using RT-qPCR

RT-qPCR reactions were performed similar as described previously (Reichelt et al., 2018). In short, total isolated RNA was reverse transcribed using the ProtoScript^® II First Strand cDNA Synthesis Kit (NEB), according to the manufacturer’s instructions and using a random primer mix (Promega). The reactions were assembled in triplicates using the qPCRBio SyGreen Mix Lo-Rox Kit (PCR Biosystems) with reverse transcribed cDNA from the first step in a 1:10 dilution, including a control reaction that lacked the reverse transcriptase (-RT) and a no template control (NTC). RT-qPCR reactions were run on a Rotor-Gene Q cycler (Qiagen) in a three-step protocol: 95°C – 10’ for one cycle; 95°C – 30”, 58°C – 30”, 72°C – 30” for 40 cycles. Data evaluation was done using the corresponding Rotor-Gene Q software package (Qiagen). Relative expression levels were calculated using the delta-delta Ct method (2^–ΔΔCt), by comparing the Ct values from biological triplicates of the gene of interest to a house-keeping gene pf0256. The applicability of pf0256 as a calibrator was evaluated before (Reichelt et al., 2018). Accordingly, all primer efficiencies were tested and only primers with efficiencies between 90 and 110% further used in the experiments.