Quantitative PCR gene expression analysis

RNA was extracted using TRIzol (Invitrogen) based on the manufacturer's instructions. DNA was removed with DNase I (New England Biolabs) and RNA was purified using Monarch® RNA Cleanup Kit (New England Biolabs). Complementary DNA (cDNA) was generated using High‐Capacity RNA‐to‐cDNA Kit (Applied Biosystems) and the cDNAs were diluted 10‐fold prior to qPCR measurements. The qPCR assay was performed with iTaq Universal SYBR Green Supermix (BioRad) on CFX384 Real‐Time System machine (BioRad). qPCR primers are listed in Appendix Table S1. All experiments were performed in technical and biological triplicates. Primer efficiency and qPCR quantification were analyzed as previously described (Siwek et al, 2020 (link)).