Quantitative RT-qPCR for Host and Viral Gene Expression

Total RNA was extracted from cell lysates using the RNeasy Plus kit (Qiagen) according to the manufacturer’s protocol and reverse-transcribed [25 (link)] with iScript^TM Reverse Transcription Supermix (Biorad, Hercules, CA, USA) using random hexamer primers. The expression levels of target genes and M1 were measured using the CFX Connect qPCR platform (BioRad) and iTaq^TM Universal SYBR^® Green Supermix (BioRad). The qPCR thermal cycle was as follows: initial denaturation 3 min at 95°C, followed by 40 cycles including 10 s at 95°C and 30 s at 60°C. Each point was performed in triplicate. To make sure that the primers produced a single and specific PCR amplification product, a dissociation curve was carried out at the end of the PCR cycle. Host genes were quantified using the comparative ΔΔCt method. The mean ΔCt obtained in mock-infected cells for each gene was used as calibrator, after normalization to endogenous HPRT, GAPDH and β-tubulin housekeeping genes. Details of the primers are provided in supplemental materials. Chicken RNA samples were processed as previously described for host response [32 (link)] and viral RNA quantifying [37 (link)].