Abstract
Due to the limitations in the range of antibodies recognising avian viruses, quantitative real-time PCR (RT-qPCR) is still the most widely used method to evaluate the expression of immunologically related genes in avian viruses. The objective of this study was to identify suitable reference genes for mRNA expression analysis in chicken intraepithelial lymphocyte natural killer (IEL-NK) cells after infection with very-virulent infectious bursal disease virus (vvIBDV). Fifteen potential reference genes were selected based on the references available. The coefficient of variation percentage (CV%) and average count of these 15 genes were determined by NanoString technology for control and infected samples. The M and V values for shortlisted reference genes (ACTB, GAPDH, HMBS, HPRT1, SDHA, TUBB1 and YWHAZ) were calculated using geNorm and NormFinder. GAPDH, YWHAZ and HMBS were the most stably expressed genes. The expression levels of three innate immune response related target genes, CASP8, IL22 and TLR3, agreed in the NanoString and RNA sequencing (RNA-Seq) results using one or two reference genes for normalisation (not HMBS). In conclusion, GAPDH and YWHAZ could be used as reference genes for the normalisation of chicken IEL-NK cell gene responses to infection with vvIBDV.
Highlights
Gene ACTB ALB B2M G6PDH GAPDH HMBS HPRT1 IFNA3 RPL4 RPL30 SDHA TBP TUBB1 UB YWHAZ
It is important to carry out an experiment to find out the best reference genes for chicken intraepithelial lymphocyte natural killer (IEL-NK) cells infected with very-virulent infectious bursal disease virus (vvIBDV) strain UPM0081
The IEL-NK cells were isolated from duodenum samples collected from uninfected chicken and chicken infected by vvIBDV at 1 and 3 dpi
Summary
Gene ACTB ALB B2M G6PDH GAPDH HMBS HPRT1 IFNA3 RPL4 RPL30 SDHA TBP TUBB1 UB YWHAZ. across different types of virus infection in the same cell type, i.e. chicken embryo cells in this case. It is important to carry out an experiment to find out the best reference genes for chicken intraepithelial lymphocyte natural killer (IEL-NK) cells infected with vvIBDV strain UPM0081. There are many new technologies that can be used for expression studies, such as NanoString and RNA-Seq technology, which allow for the detection of the expression levels of a large number of genes in a single run. NanoString and RNA-Seq have been used for reference gene selection in previous studies[10,11,12,13,14]. In this study, NanoString RNA-Seq and RT-qPCR were utilised to get a better view of the expression profile of reference genes and to identify the best reference genes
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