Validation of housekeeping genes as candidate internal references for quantitative expression studies in healthy and nervous necrosis virus-infected seven-band grouper (Hyporthodus septemfasciatus)

Rahul Krishnan,Myung-Joo Oh,Jong-Oh Kim,Jae-Ok Kim,Syed Shariq Nazir Qadiri

doi:10.1186/s41240-019-0142-3

Abstract

BackgroundIn the present study, we evaluated four commonly used housekeeping genes, viz., actin-β, elongation factor-1α (EF1α), acidic ribosomal protein (ARP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal references for quantitative analysis of immune genes in nervous necrosis virus (NNV)-infected seven-band grouper, Hyporthodus septemfasciatus.MethodsExpression profiles of the four genes were estimated in 12 tissues of healthy and infected seven-band grouper. Expression stability of the genes was calculated using the delta Ct method, BestKeeper, NormFinder, and geNorm algorithms. Consensus ranking was performed using RefFinder, and statistical analysis was done using GraphpadPrism 5.0.ResultsTissue-specific variations were observed in the four tested housekeeping genes of healthy and NNV-infected seven-band grouper. Fold change calculation for interferon-1 and Mx expression using the four housekeeping genes as internal references presented varied profiles for each tissue. EF1α and actin-β was the most stable expressed gene in tissues of healthy and NNV-infected seven-band grouper, respectively. Consensus ranking using RefFinder suggested EF1α as the least variable and highly stable gene in the healthy and infected animals.ConclusionsThese results suggest that EF1α can be a fairly better internal reference in comparison to other tested genes in this study during the NNV infection process. This forms the pilot study on the validation of reference genes in Hyporthodus septemfasciatus, in the context of NNV infection.

Highlights

The analysis and quantification of mRNA expression in different animal experimental settings are crucial in understanding the cause or outcome of a biotic or abiotic factor under study
The ideal reference gene for qPCR should have a constant expression in different tissues/cells or developmental stages and should be unaffected by the experimental situations (Radonic et al 2004)
Housekeeping genes are generally involved in the maintenance of cellular homeostasis, they are assumed to be constitutively expressed; many studies demonstrated that the expression levels of these genes vary significantly with different factors (Ingerslev et al 2006; McCurley and Callard 2008; Su et al 2011; Paria et al 2016)

Summary

Introduction

The analysis and quantification of mRNA expression in different animal experimental settings are crucial in understanding the cause or outcome of a biotic or abiotic factor under study. The quality and accuracy of the data generated from a qPCR experiment relies on the normalization of the output with a constitutively expressed gene to avoid experimental errors caused by cDNA concentration, variations in RNA, reverse transcription efficiency, and PCR efficiency (Dheda et al 2004). The ideal reference gene for qPCR should have a constant expression in different tissues/cells or developmental stages and should be unaffected by the experimental situations (Radonic et al 2004). Housekeeping genes such as those encoding βactin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and elongation factor 1 alpha (EF1α) are commonly used as internal references. We evaluated four commonly used housekeeping genes, viz., actin-β, elongation factor-1α (EF1α), acidic ribosomal protein (ARP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal references for quantitative analysis of immune genes in nervous necrosis virus (NNV)-infected seven-band grouper, Hyporthodus septemfasciatus

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