2.4. cDNA Synthesis and Reverse Transcription Quantitative PCR (RT-qPCR)

Here, 2 μg of total RNA was reverse transcribed into cDNA using GoScript Reverse Transcription System (Promega, Mannheim, Germany) according to the manufacturer’s protocol. The cDNA was diluted 1:8 with sterile water and stored at −20 °C. RT-qPCR analysis was performed using LightCycler 480 SYBR Green I master mix and the LightCycler 480 instrument (Roche Applied Science, Penzberg, Germany). The Cp value was extracted using the second derivative maximum method from the LightCycler software. The relative expression to the housekeeping genes RPS13 and RPS23 was calculated by the ΔΔCp method, including primer efficiency in the calculation [18]. Sequences of primers are listed in the Supplementary Table S1 and the experiments were repeated at least three times.