Quantitative RT-qPCR for IL10 and IL15 Expression

Quantitative RT-qPCR was carried out in a total reaction volume of 10 µL containing 1× each of TaqMan gene expression assays (20×) (human IL10 (Hs00174086_m1), and human IL15 (Hs01003716_m1), ThermoFisher Scientific), 1× TaqMan Fast Advanced Master Mix (2×) (ThermoFisher Scientific), and 10 ng of cDNA. RT-qPCR reactions were performed in a StepOnePlus Real-Time PCR Systems thermocycler (ThermoFisher Scientific), and relative expression of target genes was normalized by human GAPDH (Hs99999905_m1; Thermo Fisher Scientific), a very common reference gene used in the literature and highly standardized in our laboratory [13 (link),23 (link)]. RT-qPCR data analysis was performed with the N₀ method implemented in LinRegPCR v. 2020.0, which considers RT-qPCR mean efficiencies estimated by the window-of-linearity method [28 (link),29 (link)]. Briefly, N₀ values were calculated in LinRegPCR using default parameters. Then, N₀ values from the gene of interest (GOI) were normalized by taking their ratio to the N₀ of the reference gene GAPDH (N_0GOI/N_0REF).