Reference gene selection for gene expression study in shell gland and spleen of laying hens challenged with infectious bronchitis virus

www.nature.com/scientificreports OPEN Received: 9 May 2017 Accepted: 13 October 2017 Published: xx xx xxxx Reference gene selection for gene expression study in shell gland and spleen of laying hens challenged with infectious bronchitis virus Samiullah Khan1,2, Juliet Roberts1 & Shu-Biao Wu 1 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. The five main segments of hen oviduct are ovary, infundibulum, magnum, isthmus and shell gland (uterus). The isthmus is involved mainly in shell membrane formation. The shell gland is involved in the synthesis and secretion of substances for the formation of distinct layers of the eggshell. During the egg formation cycle, an egg remains for approximately 18–20 hours in the shell gland during which shell formation takes place1. Calcium ions for shell formation are secreted from the shell gland cells and the calbindin gene plays a primary role in Ca2+ transportation2. Approximately 437 peptides and ion transporters have been identified as being involved in the formation of the eggshell3,4. Based on the role of the shell gland in synthesis of various components the eggshell, it is, metabolically, a very active organ in the reproductive tract of laying hens. Infectious bronchitis virus (IBV) is a highly contagious mucosal pathogen of both broiler and layer chickens worldwide5,6. IBV replicates in cell cytoplasm and contains an un-segmented single stranded positive sense RNA of 27.6 kbp7–9. IBV has a short incubation period6, and viral spread occurs rapidly among chickens by aerosol and mechanical means10,11. IBV has the capability to multiply in various epithelial tissues, such as trachea12,13, kidney14, intestine15,16, spleen17 and oviduct16,18–20. The virus is well known for its effects in laying hens, including egg production and quality drops10,16,21,22. In Australia, there are two common forms of this virus, respiratory and nephropathogenic. Both types can induce various degrees of pathological changes in the oviduct of adult laying hens20. Genes involved in eggshell formation have been shown to be affected by IBV infection23. IBV infection induces a wide range of immune responses in chickens. An innate immune response is activated during the initial stages of infection in the mucosal lining of the trachea following binding of IBV virions to receptors on epithelial cells24. Activation of the innate immune response may be initiated by Toll-like receptors (TLRs) signalling upon IBV recognition25. Cellular and local immunity play a critical role in the protection of chicks from IBV infection26. Studies have shown that systemic immunisation generally fails to elicit strong mucosal immunity27,28. However, all 1 Animal Science, School of Environmental and Rural Science, University of New England, Armidale, New South Wales, 2351, Australia. 2Present address: School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, South Australia, 5371, Australia. Correspondence and requests for materials should be addressed to S.-B.W. (email: ) SciENTific REPOrTS | 7: 14271 | DOI:10.1038/s41598-017-14693-2 1 www.nature.com/scientificreports/ ages are susceptible, with very young chicks exhibiting more severe respiratory signs and much higher mortality than older birds29,30. The spleen is a lymphoid organ that plays an important role in the initiation of the immune response against systemically induced antigens31 and is among the major organs where T and B cells are localized. In birds, the spleen serves as an important secondary immune organ as lymph nodes are not present31,32. Quantifying gene expression in various patho-physiological conditions is a common technique in molecular biology. The two most commonly used methods of performing quantitative gene expression include relative and absolute quantification33. In relative quantification, qPCR data of candidate target genes of interest are achieved by including two or more most stably expressed internal control genes as an internal calibrator (reference genes)34. Selection of a reliable reference gene under the specific conditions is key to quantitative accuracy. The ideal reference gene should be expressed at a constant level in the tissue regardless of tissue nature, cell type, developmental stage and experimental conditions34,35. Traditionally, most commonly used housekeeping genes, such as ACTB, TUBB and GAPDH have been used widely as generic reference genes. However, ample evidence has shown that the expression of these genes may not be constant across a range of experimental conditions and tissues under investigation36–38. Thus, it is now recommended to use housekeeping genes as reference genes for normalisation only when prior analysis of their expression stability has been carried out. It is also recommended that more than one reference gene be used to achieve more robust, accurate and reliable normalisation of gene expression data34. The programmes geNorm34,39, NormFinder40,41 and BestKeeper42 have been used to analyse the stability of housekeeping gene expression in samples from various sources. The underlying principle in each software is slightly different from the others and thus the resulting ranking of genes is not always the same. geNorm in qbase + module version 3.0 (Biogazelle, Belgium) calculates the gene expression stability (geNorm M) as the arithmetic mean of the pairwise variation (geNorm V) between all tested genes34,39. The geNorm V for any given two genes is the standard deviation calculated from the log2 transformed relative quantities between those two genes34 geNorm V shows level of variation in the average values of reference gene stability with the sequential inclusion of the next stable reference gene to the equation (...truncated)