MIQE-Compliant qPCR Protocol for Gene Expression

qPCR experiments were performed as per the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al. 2009 (link)). qPCR was carried out in LightCycler480 II 96 multi-well plates (Roche Diagnostics, Burghess Hill, UK) using a LightCycler480 II (Roche Diagnostics, Burghess Hill, UK). Reactions were set up following the manufacturer’s instructions using SYBR® Green I master mix (Roche Diagnostics, Burghess Hill, UK), and the primer sets listed in Table S1. The qPCR cycling conditions were set at 95 °C for 5 min and 45 cycles for 30 s at 95 °C, 20 s at 60 °C, and 30 s at 72 °C. Three technical replicates of all experimental samples were analysed, and data were reported as fold change normalised to the house-keeping gene (GAPDH) relative to untreated control and computed as 2^−ΔΔCq (Maussion et al. 2021 (link)).