High green mastermix

The RNA was transcribed into cDNA (SuperScript III RT, Thermo Fisher Scientific, USA) with a final concentration of 25 ng/µL. All steps from RNA isolation to cDNA synthesis were performed in parallel for all samples of each experiment in order to avoid experimental variations. RT-qPCR was performed in technical duplicates using 2.5 ng/µL cDNA in each reaction and a primer concentration of 0.5 µM. The qTower3 (Analytik Jena, Jena, Germany), High Green Mastermix (Thermo Fisher Scientific, USA), qPCRSoft 3 (Analytik Jena, Germany) and self-designed intron spanning primers were used (Eurofins Genomics, Luxembourg). Primers were designed by using Primer-BLAST (NCBI, Bethesda, MD, USA) followed by a PCR-Check (Eurofins Oligo Analyse Tool, Luxembourg) to ensure in silico PCR specificity. RT-qPCR protocol was performed as follows: 2 min 50 °C for 2 min, 95 °C for 10 min followed by 40 cycles of 95 °C/15 s, 60 °C/30 s and 72 °C/30 s. After 95 °C for 15 s as the last step, a melting curve (60–95 °C). Gene, primer and target/amplicon information for the reference and target genes are displayed in Table 1.

The RNA was transcribed into cDNA (SuperScript III RT, Thermo Fisher Scientific, USA). Basing upon the measurement after RNA purification, the final concentration was 25 ng/µL. All steps from RNA isolation to cDNA synthesis were performed in parallel for all samples of each experiment in order to avoid experimental variations.
RT-qPCR was performed in technical duplicates using 2.5 ng/µL cDNA in each reaction and a primer concentration of 0.5 µM. The qTower^{3 (link)} (Analytik Jena, Germany), High Green Mastermix (Thermo Fisher Scientific, USA), qPCR-Soft 3 (Analytik Jena, Germany) and self-designed intron spanning primers were used (Eurofins, Luxembourg). Primers were designed by using Primer-BLAST (NCBI, USA) followed by a PCR-Check (Eurofins Oligo Analyse Tool, Luxembourg) to ensure in silico qPCR specificity. Our criteria were length ca 20 bp, annealing temperature 60 °C, max product length 200 bp, intron spanning, covering possible transcript variants. The RT-qPCR protocol included an initial step of 50 °C for 2 min, 95 °C for 10 min followed by 40 cycles of 95 °C/15 s, 60 °C/30 s and 72 °C/30 s. A step of 95 °C for 15 s forms the transition to melting curve analysis (60–95 °C).

For real-time (RT) qPCR, the intron-spanning primers (Eurofins, Luxembourg, Luxembourg) were designed by using Primer-BLAST (NCBI) and the Roche Universal Primer Library Tool (Roche Diagnostics, Mannheim, Germany), as mentioned in our work by Niederau et al. [24 (link)]. In silico qPCR specificity was examined with a PCR check (Eurofins Oligo Analysis Tool, Luxembourg) regarding intron-spanning, length (20 bp) and product length (200 bp), transcript variations and annealing temperature. Each cDNA sample was analyzed in technical duplicates using 2.5-ng/µL cDNA with a primer concentration of 0.5-µM and High Green Mastermix (Thermo Fisher Scientific, Waltham, MA, USA). Real-time (RT) qPCR was carried out with qTower3 and qPCR-Soft 3 (Analytik Jena GmbH, Jena, Germany), with a 2-min initial warming (50 °C) and heating up to 95 °C for 10 min, followed by 40 cycles of 95 °C/15 s, 60 °C/30 s and 72 °C/30 s. All primers are displayed in Table 1. Additional primer information about the gene function, acc. no., chromosomal location and primer location, amplicon length and location can be found in the Supplementary Materials Table S1.