Optimized qPCR Primer Design and Amplification

Primer design was based on the official gene nucleotide sequences from the NCBI Nucleotide database (GeneBank, National Centre for Biotechnology Information, Bethesda MD, USA). They were constructed with NCBI PrimerBLAST^{38 (link)} considering the final concentration of qPCR components according to optimized criteria^{11 (link),39 (link)–41 (link)}. Primers received no terminal or other modifications and were synthesized and purified by Eurofins MWG Operon LLC (Huntsville, AL, USA; High Purity Salt Free Purification HPSF^®). For qPCR amplification we used a Mastercycler^® ep realplex-S thermocycler (Eppendorf AG, Hamburg, Germany) in conjunction with 96 well PCR plates (TW-MT, 712282, Biozym Scientific GmbH, Hessisch Oldendorf, Germany) and BZO Seal Filmcover sheeting (712350, Biozym Scientific GmbH). Into each well SYBR^®Green JumpStart™ Taq ReadyMix™ (7.5 µl, Sigma–Aldrich^®, S4438), consisting of Tris–HCl (20 mM, pH 8.3), KCl (100 mM), MgCl₂ (7 mM), dNTPs (0.4 mM per dATP, dCTP, dGTP, dTTP), stabilizers, Taq-DNA-polymerase (0.05 U/µl), JumpStart™ Taq antibody and SYBR^®Green I, as well as the respective cDNA solution (1.5 µl, dilution 1:10) and the respective primer pair (7.5 pmol, 0.75 µl–3.75 pmol/primer) were pipetted ad 15 µl nuclease-free H₂O (T143, Carl-Roth GmbH). We amplified the cDNA in triplets (three technical replicates) per candidate reference gene and biological replicate (sample) and on the same qPCR plate in 45 cycles (initial heat activation 95 °C/5 min, per cycle 95 °C/10 s denaturation, 60 °C/8 s annealing, 72 °C/8 s extension, Supplementary Data 3), resulting in 6 (samples) × 3 (experimental conditions) × 3 (technical replicates) analysed PCR reactions (C_q values) per candidate reference gene. At the end of each extension step SYBR^®Green I fluorescence was measured at 521 nm.