Itaq universal sybr green supermix kit reagents

In our previous study, the transcriptome profiles of ALV-J-induced tumors in spleen samples compared to healthy spleen samples from White Recessive Plymouth Rock chickens were used to identify the genes related to ALV-J invasion (Li et al., 2015 (link)). In this study, the related genes of innate immune transcriptional responses were analyzed according to the transcriptome profiles.
Expression of related genes of innate immunity were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was extracted from frozen spleens of SPF chickens and clinical samples using the RNAfast200 kit (Fastagen), followed by cDNA synthesis of mRNA with the RevertAid First strand cDNA synthesis kit (Thermo-Fisher Scientific) according to the manufacturer’s instructions. qRT-PCR was performed on a Biorad CFX96 Real-Time Detection System using iTaq^TM Universal SYBR^® Green Supermix Kit reagents (Biorad, CA, USA) according to the manufacturer’s specifications. Primers used for qRT-PCR were designed using the NCBI Primer BLAST program¹ and were based on published target sequences (Table 1). Data analyses were performed using the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)).

Total RNA was extracted from ALV-J-infected (10⁵ TCID₅₀/mL) and uninfected MDM at 3 hpi and 36 hpi using RNAiso reagent (TaKaRa, Japan). For gene expression analysis, cDNA synthesis of mRNA was performed using a PrimeScript RT Reagent Kit (Perfect Real Time) (TaKaRa, Japan) according to the manufacturer’s protocol. The qPCR primers were designed using the NCBI Primer BLAST program [16 (link)] and were based on published target sequences (Additional file 1A) [17 (link)–20 (link)]. The GAPDH gene was used as an internal control. qPCR was performed on a Bio-Rad CFX96 Real-Time Detection System using iTaqTM Universal SYBR^® Green Supermix Kit reagents (Bio-Rad, CA, USA) according to the manufacturer’s specifications. Data analyses were performed using the 2^−ΔΔCt method.