Re-evaluation of common carp (Cyprinus carpio L.) housekeeping genes for gene expression studies – considering duplicated genes

Szczygieł, Joanna; Kamińska-Gibas, Teresa; Petit, Jules; Jurecka, Patrycja; Wiegertjes, G.F.; Irnazarow, Ilgiz

doi:10.1016/j.fsi.2021.05.013

Cited by 9 publications

(5 citation statements)

References 53 publications

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“…GAPDH , the least stable reference gene in the tissues of each SBM level sampled at 2 W, was ranked among the top three stable genes in the tissues of the 0% and 50% SBM levels sampled at 6 W. These results revealed that the expression stability of the six reference genes in hybrid yellow catfish varied based on the tissues, sampling time points, and SBM dosage. Many studies have reported that experimental conditions, such as dietary restriction [ 32 ], metals [ 22 ], viral infection [ 33 , 34 ], and herbicides [ 20 ], influence the expression of reference genes that have traditionally been considered as reference genes (e.g., GAPDH , HPRT1 , and 18S rRNA ). This could be attributed to each organ's different functions and heterogeneity and the characteristic reactions of the evaluated reference genes to specific conditions [ 35 ].…”

Section: Discussionmentioning

confidence: 99%

Screening and Stability Analysis of Reference Genes for Gene Expression Normalization in Hybrid Yellow Catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂) Fed Diets Containing Different Soybean Meal Levels

Guo,

Zhang,

Zhang

et al. 2023

Aquaculture Nutrition

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In this study, we screened the expression stability of six reference genes (18S rRNA, β-actin, GAPDH, EF1a, B2M, and HPRT1) in hybrid yellow catfish (n = 6), considering the SBM levels, sampling time points, and different tissues. Four different statistical programs, BestKeeper, NormFinder, Genorm, and Delta Ct, combined with a method that comprehensively considered all results, were used to evaluate the expression stability of these reference genes systematically. The results showed that SBM levels significantly impacted the expression stability of most of the reference genes studied and that this impact was time-, dose-, and tissue-dependent. The expression stability of these six reference genes varied depending on tissue, sampling time point, and SBM dosage. Additionally, more variations were found among different tissues than among different SBM levels or sampling time points. Due to its high expression, 18S rRNA was excluded from the list of candidate reference genes. β-actin and GAPDH in the liver and β-actin, HPRT1 and EF1a in the intestine were the most stable reference genes when SBM levels were considered. HPRT1, and EF1a in tissues sampled at 2 W and EF1a and β-actin in tissues sampled at 4 and 6 W were proposed as two stable reference genes when different tissues were considered. When the sampling time points were considered, β-actin, EF1a, and HPRT1 were the top three stable reference genes in the intestine. In contrast, β-actin and B2M are the most stable reference genes in the liver. In summary, β-actin, EF1a, and HPRT1 were the more stable reference genes in this study. The stability of reference genes depends on the tissues, sampling time points, and SBM diet levels in hybrid yellow catfish. Therefore, attention should be paid to these factors before selecting suitable reference genes for normalizing the target genes.

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Section: Discussionmentioning

confidence: 99%

Screening and Stability Analysis of Reference Genes for Gene Expression Normalization in Hybrid Yellow Catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂) Fed Diets Containing Different Soybean Meal Levels

Guo,

Zhang,

Zhang

et al. 2023

Aquaculture Nutrition

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“…RPL7 is a member of the ribosomal protein family, and it was considered a suitable reference gene under experimental conditions in the previous studies (Wang et al ., 2018; Penning et al ., 2007). 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). Although the stability ranking of the five candidate reference genes was different in various tissues or under experimental conditions, RPL7 and EF1A were stable in multiple A. anguilla tissues in different growth periods and in eels challenged by AngHV.…”

Section: Discussionmentioning

confidence: 99%

“…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%

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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“…These bacteria were isolated from infected carps and selected on an appropriate medium for Aeromonas (Rimler-Shotts Agar) by the Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture in Golysz in Poland. This strain has already been used in previous studies and has been shown to cause immunological changes ( 55 – 57 ).…”

Section: Methodsmentioning

confidence: 99%

Erythrocytes of the common carp are immune sentinels that sense pathogen molecular patterns, engulf particles and secrete pro-inflammatory cytokines against bacterial infection

Majstorović,

Kyslík,

Klak

et al. 2024

Front. Immunol.

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IntroductionRed blood cells (RBCs), also known as erythrocytes, are underestimated in their role in the immune system. In mammals, erythrocytes undergo maturation that involves the loss of nuclei, resulting in limited transcription and protein synthesis capabilities. However, the nucleated nature of non-mammalian RBCs is challenging this conventional understanding of RBCs. Notably, in bony fishes, research indicates that RBCs are not only susceptible to pathogen attacks but express immune receptors and effector molecules. However, given the abundance of RBCs and their interaction with every physiological system, we postulate that they act in surveillance as sentinels, rapid responders, and messengers.MethodsWe performed a series of in vitro experiments with Cyprinus carpio RBCs exposed to Aeromonas hydrophila, as well as in vivo laboratory infections using different concentrations of bacteria.ResultsqPCR revealed that RBCs express genes of several inflammatory cytokines. Using cyprinid-specific antibodies, we confirmed that RBCs secreted tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). In contrast to these indirect immune mechanisms, we observed that RBCs produce reactive oxygen species and, through transmission electron and confocal microscopy, that RBCs can engulf particles. Finally, RBCs expressed and upregulated several putative toll-like receptors, including tlr4 and tlr9, in response to A. hydrophila infection in vivo.DiscussionOverall, the RBC repertoire of pattern recognition receptors, their secretion of effector molecules, and their swift response make them immune sentinels capable of rapidly detecting and signaling the presence of foreign pathogens. By studying the interaction between a bacterium and erythrocytes, we provide novel insights into how the latter may contribute to overall innate and adaptive immune responses of teleost fishes.

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Re-evaluation of common carp (Cyprinus carpio L.) housekeeping genes for gene expression studies – considering duplicated genes

Cited by 9 publications

References 53 publications

Screening and Stability Analysis of Reference Genes for Gene Expression Normalization in Hybrid Yellow Catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂) Fed Diets Containing Different Soybean Meal Levels

Screening and Stability Analysis of Reference Genes for Gene Expression Normalization in Hybrid Yellow Catfish (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂) Fed Diets Containing Different Soybean Meal Levels

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

Erythrocytes of the common carp are immune sentinels that sense pathogen molecular patterns, engulf particles and secrete pro-inflammatory cytokines against bacterial infection

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