Evaluation of candidate reference genes for quantitative RTqPCR analysis in goldfish (Carassius auratus L.) in healthy and CyHV-2 infected fish

Dharmaratnam, Arathi; Sudhagar, Arun; Nithianantham, Sundar Raj; Das, Satyajit; Swaminathan, T. Raja

doi:10.1016/j.vetimm.2021.110270

Cited by 17 publications

(12 citation statements)

References 38 publications

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“…EF1A, a factor for protein translation, has been used as reference genes in a variety of aquatic animals due to its constant expression during various pathogenic infections (Dharmaratnam et al, 2021;Matejusova et al, 2010;Snow et al, 2006;Szczygieł et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

“…NormFinder is based on a variance estimation approach and gives the stability values, with the lower values indicating more stability. Finally, RefFinder (http://blooge.cn/RefFinder/) was used to rank the examined housekeeping genes based on the results of different algorithms; it calculates the geometric mean of each gene, with a lower value indicating a more stable expression level (Dharmaratnam et al ., 2021).…”

Section: Methodsmentioning

confidence: 99%

“…Theoretically, reference genes should be universally expressed and unaffected under experimental conditions (Radonić et al ., 2004; Suzuki et al ., 2000a). Housekeeping genes such as glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), 18 S ribosomal RNA (18 S), elongation factor 1α (EF1A), ribosomal protein L7 (RPL7) and β‐actin (ACTB) are constitutively expressed and are often used as reference genes (Dharmaratnam et al ., 2021; Wang et al ., 2018; Huggett et al ., 2005; Thellin et al ., 1999; Vandesompele et al ., 2002). Nonetheless, the transcript levels of these genes may vary considerably across different tissues, developmental stages and health conditions (Beekman et al ., 2011; Maroufi et al ., 2010; Suzuki et al ., 2000b).…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of housekeeping genes as references for quantitative real‐time PCR analysis of European eel, Anguilla anguilla

Chen

Yang

et al. 2022

Journal of Fish Biology

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Eels are important aquaculture species for which an increasing number of reference genes are being identified and applied. In this study, five housekeeping genes [RPL7 (ribosomal protein L7), 18 S (18 S ribosomal RNA), EF1A (elongation factor 1α), ACTB (β‐actin) and GAPDH (glyceraldehyde‐3‐phosphate dehydrogenase)] were chosen to evaluate their reliability as reference genes for quantitative real‐time PCR (qPCR) for the study of Anguilla anguilla. The expression of the selected genes in different eel tissues was determined using qPCR at different growth stages or upon challenge by Anguillid herpesvirus (AngHV), and the expression levels of these genes were then compared and evaluated using the geNorm and NormFinder algorithms. Then, RefFinder was used to comprehensively rank the examined housekeeping genes. Interestingly, the expression of the evaluated housekeeping genes exhibited tissue‐dependent and treatment‐dependent variations. In different growth periods A. anguilla tissues, the most stable genes were the following: ACTB in mucus; 18 S in skin and kidney; RPL7 in muscle, gill, intestine and brain; EF1A in heart and liver; and GAPDH in spleen. In contrast, in AngHV‐challenged A. anguilla tissues, the most stable genes were the following: 18 S in mucus; RPL7 in skin, gill, heart, spleen, kidney and intestine; EF1A in muscle and liver; and ACTB in brain. Further comparison analysis indicated that the expression of RPL7 and EF1A was stable in multiple A. anguilla tissues in different growth periods and in eels challenged by AngHV. Nonetheless, the expression level of GAPDH in eel tissues was lower, and it was unstable in several tissues. These results indicated that the selection of reference genes for qPCR analysis in A. anguilla should be made in accordance with experimental parameters, and both RPL7 and EF1A could be used as reference genes for qPCR study of A. anguilla at different growth stages or upon challenge by AngHV. The reference genes identified in this study could improve the accuracy of qPCR data and facilitate further studies aimed at understanding the biology of eels.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of housekeeping genes as references for quantitative real‐time PCR analysis of European eel, Anguilla anguilla

Chen

Yang

et al. 2022

Journal of Fish Biology

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“…All the cycles were performed simultaneously with reference gene, β-actin in the same plate in triplicates. β-Actin was chosen as a reference gene after testing several other potential reference genes such as 18S ribosomal RNA, glyceraldehyde-3P-dehydrogenase and elongation factor 1 alpha (Dharmaratnam et al 2021). In the present study too, the Cq values of β-actin gene did not change significantly (P > 0.05, ranged between 11 and 12).…”

Section: Gene Expression Analysismentioning

confidence: 99%

“…While a lot of studies on CyHV-2 infection in goldfish were conducted, however, there are only few reports on the host-pathogen interaction. Increased expressions of many immune-related genes were evident in crucian carp (C. carassius) to CyHV-2 infection such as intelectin, interleukin-11 (IL-11), purine nucleoside phosphorylase 5a (PNP5a), keratin8, myeloid-specific peroxidase (MPO), dual specificity phosphatase 1 (dusp1), MHC-I, IRF3 and MAPK7 (Xu et al 2014(Xu et al , 2016Lu et al 2017).…”

Section: Introductionmentioning

confidence: 99%

Immune gene expression in cyprinid herpesvirus-2 (CyHV-2)–sensitized peripheral blood leukocytes (PBLs) co-cultured with CyHV-2-infected goldfish fin cell line

et al. 2021

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Goldfish is one of the preferred ornamental fish which is highly susceptible to cyprinid herpesvirus-2 (CyHV-2) infection. The present study aimed to analyse immune gene expression in a co-culture of CyHV-2-sensitized goldfish peripheral blood leukocytes (PBLs) with CyHV-2-infected fantail goldfish fin cell lines (FtGF). Goldfish were sensitized with intraperitoneal TCID 50 dose (10 7.8±0.26 /mL) of CyHV-2. After 2 weeks, PBLs were collected and co-cultured with CyHV-2-infected FtGF cells keeping both uninfected FtGF cells and PBL control groups. After 2 days of co-culture, WST-1 assay for cell proliferation was performed at 450 nm during the 2nd, 4th and 6th days of coculture. The results showed a significant increase (p < 0.05) in cell density in CyHV-2infected PBL and virus-infected FtGF cells during the 4th day post co-culture which confirmed effector cell generation. Expressions of few immune genes were checked taking RNA samples of CyHV-2-induced PBLs post co-culture with infected FtGF cells along with uninfected FtGF cells as control group at different time periods (2nd, 4th and 6th days) in triplicate. The results indicated increased expression of CD8α, IFNγ, b2m, MHC I, LMP 7, IL-10, IL-12 and GATA3 except Tapasin. From the above study, we concluded that goldfish showed both Th1-and Th2-mediated immune responses to CyHV-2. The current findings support the scope for further vaccine development against CyHV-2 for goldfish.

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Biotic and abiotic stress affects the stability of housekeeping genes in Catla Labeo catla

Acharya,

Tripathi,

Rasal

et al. 2024

J Aqua Anim Hlth

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ObjectiveThe choice of a stable reference gene plays a crucial role in gene expression analysis. In most cases, the choice of housekeeping gene (HKG) is quite random. However, the expression of HKGs varies under different experimental conditions and in different tissues.MethodsThe study focused on three reference genes in Catla Labeo catla—beta actin (β‐actin), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), and elongation factor‐1 alpha (EF‐1α)—under four experimental conditions: bacterial challenge, hypoxia, a combination of bacterial challenge and hypoxia, and control. The fish were subjected to the above‐mentioned experimental circumstances, and five tissues (blood, gill, liver, kidney, and spleen) were collected at regular intervals.ResultThe investigation of gene expression in the five tissues was performed using real‐time (RT) quantitative polymerase chain reaction (qPCR), and the resultant cycle threshold values were subjected to several statistical techniques via the RefFinder tool to determine the most stable HKG. The results were ordered in accordance with the stability of the reference genes. In this study, GAPDH was found to be the least stable gene, while β‐actin and EF‐1α remained stable in the kidney and liver under various circumstances. Other tissues demonstrated tissue‐ and treatment‐specific stability for the various HKGs.ConclusionThe findings indicate that GAPDH should be avoided in Catla for gene expression studies and that different algorithms should be employed to validate the stability of HKGs in RT‐qPCR analysis.

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Evaluation of candidate reference genes for quantitative RTqPCR analysis in goldfish (Carassius auratus L.) in healthy and CyHV-2 infected fish

Cited by 17 publications

References 38 publications

Evaluation of housekeeping genes as references for quantitative real‐time PCR analysis of European eel, Anguilla anguilla

Evaluation of housekeeping genes as references for quantitative real‐time PCR analysis of European eel, Anguilla anguilla

Immune gene expression in cyprinid herpesvirus-2 (CyHV-2)–sensitized peripheral blood leukocytes (PBLs) co-cultured with CyHV-2-infected goldfish fin cell line

Biotic and abiotic stress affects the stability of housekeeping genes in Catla Labeo catla

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