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

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

doi:10.1186/s41240-019-0142-3

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

Fisheries and aquatic sciences, v.22 no.12, 2019년, pp.28.1 - 28.8

Krishnan, Rahul (Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University) , Qadiri, Syed Shariq Nazir (Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University) , Kim, Jong-Oh (Institute of Marine Biotechnology, Pukyong National University) , Kim, Jae-Ok (National Institute of Fisheries Science) , Oh, Myung-Joo (Department of Aqualife Medicine, College of Fisheries and Ocean Science, Chonnam National University)

Abstract ▼ AI-Helper

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 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. 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.

주제어

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문제 정의

The identification of a suitable reference gene in seven-band grouper is crucial for accurate immune gene expression profiling. The objective of the current study was to validate housekeeping genes of seven-band grouper to identify a candidate reference gene as an internal control for expression profiling studies during NNV infection.

제안 방법

RK and OMJ designed this study and wrote the manuscript. RK, SSNQ, JOK, and JOK performed the experiments and analyzed the data.
RK and OMJ designed this study and wrote the manuscript. RK, SSNQ, JOK, and JOK performed the experiments and analyzed the data. All authors read and approved the final manuscript.
To evaluate the performance of housekeeping genes in the fold change evaluation of immune genes, expression of IFN-1 and Mx in the brain, eye, spleen, kidney, and blood was analyzed (Fig. 5). Significant variation in the fold change expression and calculation thereof was observed in the tissues using different housekeeping genes.
To further evaluate the expression stability of housekeeping genes, a comparative fold change expression analysis of immune genes, IFN-1 and Mx, at 48 h postinfection was undertaken. Fold change was separately calculated for both immune genes with actin- β, EF1α, ARP, and GAPDH as internal controls using the comparative CT method (2^−ΔΔCT method) (Livak and Schmittgen 2001).

데이터처리

Statistical analysis for the differences in the expression level of actin-β, EF1α, ARP, and GAPDH was done by linear regression and two-way analysis of variance (ANOVA) using GraphPad Prism 5.0 (La Jolla, CA, USA).

이론/모형

, actin- β, elongation factor-1α (EF1α), acidic ribosomal protein (ARP), and glyceraldehyde 3- phosphate dehydrogenase (GAPDH). Specific primers for each of the genes were designed based on the nucleotide sequences from the H. septemfasciatus brain transcriptome data (Kim et al. 2017) using Primer-BLAST suite (https:// www.ncbi.nlm.nih.gov/tools/primer-blast/) (Table 1). The optimal annealing temperature of each primer was about 60 °C and the amplicons were within the range of 100–150 bp.

성능/효과

In conclusion, the expression and stability of the four housekeeping genes tested in this study have tissuedependent variation in healthy as well as NNV-infected fish seven-band grouper tissues with significant variation in the fold change calculations for immune genes. Considering the consensus ranking of overall stability, EF1α was found to be the most suitable reference gene in infection studies of grouper.
Significant variation in the expression of all the four genes was observed in the spleen while the expression of GAPDH, actin-β, and ARP was variable in the liver.
Statistical analysis showed a significant difference in the expression among all genes and also with time for ARP, actin-β, and GAPDH.

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