One step sybr green master mix 2

For relative quantification of vRNA and cytokine mRNA in CEFs, total RNA was extracted from virus-infected CEFs samples by using RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. The vRNA and IFN-stimulated genes (ISGs) were then quantified by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) in a thermal cycler (Smart Cycler System, Cepheid, Sunnyvale, CA, USA) using One-Step SYBR Green Master Mix II (Takara, Dalian, Jiangsu, China) according to the manufacturer’s instructions. The primer sets for hemagglutinin (HA), polymerase basic 2 (PB2) genes, chicken IFN-α, chicken IFN-β, chicken myxovirus resistant 1 (chMx), chicken 2'-5' oligoadenylate synthetase (chOAS), chicken RNase L, and chicken protein kinase R (chPKR) mRNAs are listed in Additional file 1: Table S1. The vRNA was quantified and compared to that in the mock-transfected control. The expression of cytokines and ISGs was normalized using the comparative 2^-2∆∆Ct method, which was used to determine the mean fold increase in the expression level of the respective gene from the corresponding time point in the uninfected cell [21 (link)].

Total RNAs were extracted using the RNeasy Mini Kit (Qiagen, USA) according to the manufacturer’s instructions. Expression of the TLR3, TLR7, TLR8, RIG-I, and MDA5 mRNAs was determined by RT-qPCR using the PrimeScript RT Reagent Kit (Takara, Japan) according to the manufacturer’s instructions (primer and probe sequences listed in Table 1). Expression levels of the IRF3, IRF7, and IFN-β mRNAs were estimated by RT-qPCR using One-Step SYBR Green Master Mix II (Takara) according to the manufacturer’s instructions (primer sequences listed in Table 2). The relative changes in mRNA expression levels of H3N2 CIV-infected KU-CBE cells to those of uninfected control cells were determined at various time points using the comparative 2^-ΔΔCt method [20 (link)].

Three innate immune-related genes in splenic total RNA were quantified by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) in a thermal cycler (Smart Cycler System, Cepheid, Sunnyvale, CA, USA) using a One-Step SYBR Green Master Mix II (Takara, Dalian, Jiangsu, China) according to the manufacturer’s instructions. Primer sets for quantification of chicken C-type lectin (chC-lectin), chicken 2′-5′-oligoadenylate synthetase-like (chOASL), chicken myxovirus resistant 1 (chMx), and chicken GAPDH (chGAPDH) mRNAs are listed in Table S1. The transcription level of mRNA was normalized using the comparative 2^−ΔΔCt method, which was used to determine the mean fold increase in the expression level of the respective genes from corresponding uninfected chickens [17 (link)].