Abstract
Ten reference genes were investigated for normalisation of candidate target gene expression data in the shell gland and spleen of laying hens challenged with two strains of infectious bronchitis virus (IBV). Data were analysed with geNorm, NormFinder and BestKeeper, and a comprehensive ranking (geomean) was calculated. In the combined data set of IBV challenged shell gland samples, the comprehensive ranking showed TATA-box binding protein (TBP) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) as the two most stable, and succinate dehydrogenase complex flavoprotein subunit A (SDHA) and albumin (ALB) as the two least stable reference genes. In the spleen, and in the combined data set of the shell gland and spleen, the two most stable and the two least stable reference genes were TBP and YWHAZ, and ribosomal protein L4 (RPL4) and ALB, respectively. Different ranking has been due to different algorithms. Validation studies showed that the use of the two most stable reference genes produced accurate and more robust gene expression data. The two most and least stable reference genes obtained in the study, were further used for candidate target gene expression data normalisation of the shell gland and spleen under an IBV infection model.
Highlights
The five main segments of hen oviduct are ovary, infundibulum, magnum, isthmus and shell gland
We investigated the stability of ten reference genes in the shell gland region of the oviduct in laying hens when the egg was in the isthmus (5 hr post-oviposition time-point) region of oviduct in Infectious bronchitis virus (IBV) T and Vic S strains challenged groups
The current study provides information on the expression stability of these candidate reference genes and most stably expressed reference genes are suggested for the normalisation of gene expression data in the chicken shell gland and spleen in infectious bronchitis study models
Summary
The five main segments of hen oviduct are ovary, infundibulum, magnum, isthmus and shell gland (uterus). The current study investigated the expression stability of ten commonly used reference genes in laying hens infected with IBV T and Vic S strains. This study was performed in conjunction with a broader study in which the effect of IBV on the genes involved in eggshell formation in the shell gland and immune response in the spleen was investigated. The reference genes selected were used for gene expression data normalisation of candidate target genes in the shell gland and spleen in an IBV model. Different candidate target genes involved in calcium transportation (CALB1) across cell membrane[43] and protoporphyrin synthesis (ABCB6)[44] in the shell gland and genes (IFNγ and IL7) involved in immune system in spleen of laying hens were used for the validation of the reference genes.
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