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. 2017 Oct 27;7(1):14271.
doi: 10.1038/s41598-017-14693-2.

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

Samiullah Khan  1   2 , Juliet Roberts  1 , Shu-Biao Wu  3
Affiliations

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

Samiullah Khan et al. Sci Rep. .

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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Amplification of the genes fragments from the shell gland tissue of chicken to assess the specificities of the primers used in the current study. (L) DNA ladder (bp); (1) 18 S rRNA (63 bp); (2) ALB (197 bp); (3) ACTB (139 bp); (4) GAPDH (66 bp); (5) HMBS (131 bp); (6) HPRT1 (245 bp); (7) RPL4 (235 bp); (8) SDHA (126 bp); (9) TBP (147 bp); (10) YWHAZ (61 bp); (11) ND4-positive control (137 bp); (12) TLR7-positive control (200 bp). The upper (purple) and lower (green) markers act as internal standards and are used to align the ladder analysis with the individual DNA sample analysis. The standard curve (plotting migration time against DNA amplicon size), in conjunction with the markers, is then used to calculate DNA fragment sizes for each well from the migration times measured (see Agilent 2100 Bioanalyzer Users Guide for Molecular Assays). The DNA gel in Agilent 2100 Bioanalyzer was performed as per manufacturer’s instructions of Agilent DNA 1000 Kit.
Figure 2
Figure 2
Melting curves of 10 reference genes in the shell gland samples. All melting curves showing a single peak indicated that all primers were specific in amplifying fragments of the genes and chances for primer dimers were minimum in the qPCR products. A melting phase at a ramp from 50 °C to 99 °C at 1 °C increments in Rotor-Gene Q was performed to assess the specificity of PCR amplification.
Figure 3
Figure 3
Effect of IBV challenge on the expression stability of 10 reference genes in the shell gland and spleen. Mean Cq values of the control, IBV T and Vic S strains challenged groups in the shell gland (a) and in the spleen (b). A square across the box is depicted as the median. The box indicates the 25th and 75th percentiles and the whiskers caps represent the maximum and minimum values (c) Pairwise variation (geNorm V) of genes in the shell gland (control, IBV T and Vic S strains challenge groups together), in the spleen (control, IBV T andVic S strains challenge groups together) and in the combined data set of the shell gland and spleen. In qbase + software, geNorm V was calculated as standard deviation of the log2 transformed relative quantities between those two genes. The geNorm V analysis started with the two most stably expressed genes being compared to the pair including the third (V2/3), and the process continued until the least stable gene was added (i.e. V9/10).
Figure 4
Figure 4
Relative expression levels of the candidate target genes affected by IBV T challenge in the shell gland and spleen of laying hens. (a) CALB1 expression level in the shell gland normalized with the most stable genes YWHAZ and TBP (P = 0.6783). (b) CALB1 expression level in the shell gland normalized with the least stable genes SDHA and ALB (P = 0.7788). (c) ABCB6 expression level in the shell gland normalized with the most stable genes YWHAZ and TBP (P = 0.0152). (d) ABCB6 expression level in the shell gland normalized with the least stable genes SDHA and ALB (P = 0.0713). (e) IFNγ expression level in the spleen normalized with the most stable genes YWHAZ and TBP (P = 0.0021). (f) IFNγ expression level in the spleen normalized with the least stable genes RPL4 and ALB (P = 0.0050). (g) IL7 expression level in the spleen normalized with the most stable genes YWHAZ and TBP (P = 0.0333). (h) IL7 expression level in the spleen normalized with the least stable genes RPL4 and ALB (P = 0.1130). Values are relative expression quantities and error bars show standard error. Across the treatment, a,bshow significant difference (p < 0.05). For the candidate target genes, normalised relative quantities (NRQ) were calculated in qbase + based on (2−ΔΔCq) approach with a genes specific amplification efficiencies to show the relative expression of Cq levels in folds to the mean Cq of all samples of the genes. NRQ values were further analysed in Statview software (SAS) and Tukey-Kramer test was used to differentiate level of significance (p < 0.05) between means. A total of 20 samples for each of the groups (IBV T and control) in each tissue (shell gland or spleen) were processed for qPCR assay.
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