Quantitative Real-Time PCR Analysis

RNA from the cells was isolated using the miRNeasy Mini Kit (Qiagen, Germantown, MD, USA), as mentioned above. TaqMan PCR was performed as previously described [36 (link)]. Two microliters of five-times diluted cDNA were used for PCR using the TaqMan™ Universal PCR Master Mix with FAM-labeled gene-specific probes (Thermo Fisher Scientific) in duplicate on a QuantStudio 7 thermal cycler (Thermo Fisher Scientific).
The PCR reaction was carried out as follows:
ComponentAmountTaqMan™ Fast Advanced Master Mix (2×)5 µLTaqMan™ Assay (20×)0.5 µLNuclease-free water2.5 µLcDNA2 µLThe reaction was held at 95 °C for 20 s and with 40 cycles of denaturation at 95 °C for 1 s and annealing/extension at 62 °C for 20 s. The fold change was calculated by subtracting the Ct values of the candidate gene from the reference gene (delta Ct method). GAPDH served as the reference gene, and the fold change was presented with respect to experimental controls. A list of the TaqMan assays is shown in Table 1.