Quantitative Real-Time PCR for mRNA Analysis

RNA extraction was performed using the NucleoSpin RNA isolation kit (Macherey–Nagel). cDNA was synthesized from 1 μg total RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Each cDNA sample was pre-diluted 1:20 and 4 μl were used with 4 × Luminaris Color HiGreen qPCR Master Mix (Thermo Fisher Scientific) and 250 nM primer for the qPCR reaction. A two-step cycling protocol was performed as follows: UDG pre-treatment for 2 min (50 °C), initial denaturation for 10 min (95 °C), 40 cycles of 15 s at 95 °C, 40 s at 60 °C (CFX Connect Thermal Cycler, Bio-Rad). The relative quantity of the target mRNA was normalized to beta-2-microglobulin). The fold changes in RNA expression were calculated using the 2^−ΔΔCt method^{28 (link)}. Primer sequences are listed in supplementary information.