4.5. qPCR

Stimulation and RNA isolation was performed as described for mRNA sequencing. First, 1 µg RNA was subjected to cDNA generation using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) according to the instructions provided by the manufacturer. Next, 20 ng cDNA was used in the PCR reaction with 1.5 µL of the appropriate primer (QuantiTect Primer Assay, Qiagen) and 12.5 µL of a ready-to-use qPCR master mix (QuantiTect SYBR Green PCR Kit, Qiagen). The thermal cycling program was composed of initial denaturation at 95 °C for 15 min, 39 cycles at 95 °C for 15 s, 30 s at 54 °C, and 30 s at 72 °C. Duplicates for each data point were measured. The gene for β-actin was used as internal control and the 2^−ΔΔCt method [37] for relative quantification of gene expression.