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

다국어 초록 (Multilingual Abstract)

Background: In 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.
Methods: Expression 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.
Results: Tissue-specific variations were observed in the four tested housekeeping genes of healthy and NNVinfected 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.
Conclusions: These 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.

참고문헌 (Reference)

1 김춘섭, "능성어 양식장에서의 viral nervous necrosis (VNN) 발생양상" 한국어병학회 25 (25): 111-116, 2012

2 Li Z, "β-Actin is a useful internal control for tissue-specific gene expression studies using quantitative real-time PCR in the half-smooth tongue sole Cynoglossus semilaevis challenged with LPS or Vibrio anguillarum" 29 (29): 89-93, 2010

3 Xie F, "miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs" 80 (80): 75-84, 2012

4 Tang R, "Validation of zebrafish(Danio rerio)reference genes for quantitative real-time RT-PCR normalization" 39 (39): 384-390, 2007

5 Jorgensen SM, "Validation of reference genes for real-time polymerase chain reaction studies in Atlantic salmon" 8 (8): 398-408, 2006

6 Dheda K, "Validation of housekeeping genes for normalizing RNA expression in real-time PCR" 37 (37): 112-119, 2004

7 Meng QW, "Systematics of fish" China Agriculture Press 606-622, 1995

8 Fernandes JM, "Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development" 150 (150): 23-32, 2008

9 Silver N, "Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR" 7 (7): 33-, 2006

10 Wang H, "Selection and evaluation of new reference genes for RT-qPCR analysis in Epinephelus akaara based on transcriptome data" 12 (12): e0171646-, 2017

11 Huggett J, "Real-time RT-PCR normalisation; strategies and considerations" 6 (6): 279-, 2005

12 Bustin SA, "Quantitative real-time RT-PCR-a perspective" 34 (34): 597-601, 2005

13 Andersen CL, "Normalization of real-time quantitative reverse transcription-PCR data : a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets" 64 (64): 5245-5250, 2004

14 Tang X, "Molecular cloning, tissue distribution and expression profiles of thyroid hormone receptors during embryogenesis in orange-spotted grouper (Epinepheluscoioides)" 159 (159): 117-124, 2008

15 Luo YS, "Molecular cloning and mRNA expression pattern of Sox9 during sex reversal in orange-spotted grouper (Epinepheluscoioides)" 306 (306): 322-328, 2010

16 Casadei R, "Identification of housekeeping genes suitable for gene expression analysis in the zebrafish" 11 (11): 271-276, 2011

17 Liu Q, "Homologues of sox8 and sox10 in the orange-spotted grouper Epinepheluscoioides: sequences, expression patterns, and their effects on cyp19a1a promoter activities in vitro" 163 (163): 86-95, 2012

18 Radonić A, "Guideline to reference gene selection for quantitative real-time PCR" 313 (313): 856-862, 2004

19 Heemstra, P.C., "FAO species catalogue. Vol. 16. Groupers of the world. (Family Serranidae, Subfamily Epinephelinae). An annotated and illustrated catalogue of the grouper, rockcod, hind, coral grouper and lyretail species known to date" 125-, 1993

20 Ingerslev HC, "Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.)" 43 (43): 1194-1201, 2006

21 Huang W, "Expression pattern, cellular localization and promoter activity analysis of ovarian aromatase (Cyp19a1a) in protogynous hermaphrodite red-spotted grouper" 307 (307): 224-236, 2009

22 Olsvik PA, "Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon" 6 (6): 21-, 2005

23 Øvergård AC, "Evaluation of potential reference genes for real time RT-PCR studies in Atlantic halibut (HippoglossusHippoglossus L.); during development, in tissues of healthy and NNV-injected fish, and in anterior kidney leucocytes" 11 (11): 36-, 2010

24 Su J, "Evaluation of internal control genes for qRT-PCR normalization in tissues and cell culture for antiviral studies of grass carp(Ctenopharyngodonidella)" 30 (30): 830-835, 2011

25 Purohit GK, "Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of gene expression in the murrel Channa striatus under high-temperature stress" 42 (42): 125-135, 2016

26 Zheng WJ, "Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder(Paralichthys olivaceus)" 30 (30): 638-645, 2011

27 Paria A, "Evaluation of candidate reference genes for quantitative expression studies in Asian seabass(Latescalcarifer)during ontogenesis and in tissues of healthy and infected fishes" 54 : 597-605, 2016

28 Mitter K, "Evaluation of candidate reference genes for QPCR during ontogenesis and of immune-relevant tissues of European seabass (Dicentrarchuslabrax)" 153 (153): 340-347, 2009

29 Pfaffl MW, "Determination of stable housekeeping genes, differentially regulated target genes and sample integrity : BestKeeper-Excel-based tool using pair-wise correlations" 26 (26): 509-515, 2004

30 Kim JO, "Characterization of the transcriptome and gene expression of brain tissue in sevenband grouper (Hyporthodusseptemfasciatus) in response to NNV infection" 8 (8): 31-, 2017

31 McCurley AT, "Characterization of housekeeping genes in zebrafish : male-female differences and effects of tissue type, developmental stage and chemical treatment" 9 (9): 102-, 2008

32 Livak KJ, "Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method" 25 : 402-408, 2001

33 Vandesompele J, "Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes" 3 (3): 0034-0031, 2002

34 Bustin SA, "Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays" 25 (25): 169-193, 2000