Lung RNA Extraction and Cytokine qRT-PCR

Lungs were subjected to total RNA extraction using RNAeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Total RNA concentration was measured using Nanodrop. Then, the RT of total RNA (200 ng) was performed using the Revert Aid First Strand cDNA Kit (Thermo Fisher Scientific) using random hexamers according to the manufacturers’ instructions and stored at −20 °C.
Quantitative real-time PCR (qRT-PCR) was performed using a Maxima SYBR Green qPCR Master Kit (2x) (Thermo Fisher Scientific). Sequences of selected cytokine primers are listed in Table 1. Melting curves were set post the end of the last PCR cycle. The analysis of relative expression was performed using β-Actin as the housekeeping control gene using the 2^(−ΔΔCT) equation.
ΔΔCT of vaccinated groups (at 6 wpv) = ΔCT (vaccination, challenge infection) − ΔCT (vaccination), where ΔCT (vaccination) = CT_V (target gene) − CT_V (B-Actin), and ΔCT (vaccination, challenge infection) = CT_VI (target gene) − CT_VI (B-Actin).
ΔΔCT of control infection groups = ΔCT (Control infection) − ΔCT (Control non-infected/non-vaccinated) [49 (link),50 ]. Viral RNA shedding was also measured in the lungs by qRT-PCR to calculate RNA copy numbers.