2.11. mRNA and miRNA Expression Analyses

Quantitative real-time polymerase chain reaction (qRT-PCR) analysis using a LightCycler 480 Instrument II system (Roche, Basel, Swiss) was performed as previously described [7]. The qRT-PCR experiments were conducted with three biological and three technical replicates per donor-derived primary cell culture, unless otherwise stated. The intron-spanning primer sequences used are listed in [7] and Table 1. The geometric mean of the expression levels of the housekeeping genes SDHA, RPL13A and B2M were calculated for expression normalization. The relative target gene mRNA expressions were determined by the ΔΔC_T method considering the PCR efficiency [43]. All normalized gene expression levels shown were referred to the respective target gene expression of one primary cell culture sample for the relative comparison of multiple biological samples per experiment and figures.

Additional primer sequences and annealing temperatures (T_A) used for the qRT-PCR analysis.

The TaqMan advanced miRNA Assay (Thermo Fisher Scientific; Waltham, Massachusetts, United States) was performed, according to the manufacturer’s instructions, for the miRNA expression analyses. The primer assays miRNA-21 (477975_mir) and miRNA-191 (477952_mir) were used. The expression level of miRNA-21 determined was normalized to internal housekeeping gene expression of miRNA-191 via the ΔΔC_T method.