Chemokine/cytokine induction assay by qRT-PCR

HD11 cells and primary monocytes were stimulated with various concentrations of the cNK-lysin peptides in a range of 0.1 to 100 μg/ml or with medium alone. RNA was isolated from the HD11 cells or the primary monocytes using the RNeasy Isolation Kit (Qiagen, USA), as per the manufacturer’s instructions, treated with RNase-free DNase (Qiagen) and eluted in RNase-free water (Qiagen). The concentration and purity of the RNA were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using random hexamer primers and a QuantiTect Reverse Transcription Kit (Qiagen). Real-time RT-PCR was performed using a Stratagene Mx3000P (Agilent Technologies, USA) with a QuantiTect SYBR Green PCR Kit (Qiagen) and the various chicken chemokine and cytokine primers listed in Table 1. A melting curve was obtained at the end of each run to verify the presence of a single amplification product without primer dimers. Standard curves were generated using serial, 5-fold dilutions of cDNA. The fold changes in each transcript were normalized to β-actin and are relative to the transcript expression in unstimulated cells (normalized to 1) using the comparative ΔΔCt method as previously described⁴⁵.