Reference gene selection for qPCR in mussel, Mytilus edulis, during gametogenesis and exogenous estrogen exposure

Cubero-Leon, Elena; Ciocan, Corina; Minier, Christophe; Rotchell, Jeanette M.

doi:10.1007/s11356-012-0772-9

Cited by 62 publications

(32 citation statements)

References 21 publications

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“…Some studies have been recently published to find the best reference genes for qPCR analysis in bivalves (Araya et al 2008;Dheilly et al 2011;Cubero-Leon et al 2012;Mauriz et al 2012;Siah et al 2012;Du et al 2013) and showed that for each species, tissue, challenge and even reproductive state the optimal reference gene may change. Although it is known that the selection of an inappropriate reference gene could alter the results, no routine analysis to find the most stable gene are usually made (Bustin et al 2009).…”

Section: Discussionmentioning

confidence: 99%

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Evaluation of reference genes ofMytilus galloprovincialisandRuditapes philippinaruminfected with three bacteria strains for gene expression analysis

Moreira

Pereiro

Costa

et al. 2014

Aquat. Living Resour.

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-Quantitative real-time polymerase chain reaction (qPCR) is probably the most used method for gene expression quantification because of its high sensitivity and specificity. Nevertheless, this technology can undergo experimental errors and variations. Normalization of the results using a reference gene is therefore necessary to minimize these variations. As the study of immune genes in bivalve mollusks has increased in the last years, the establishment of adequate and stable reference genes for bivalves is strongly required. We analyzed the behavior of four putative reference genes: ribosomal RNA 18S, actin, elongation factor 1 − α and α-tubulin. The suitability of these four genes as internal control for qPCR was evaluated in mussel (Mytilus galloprovincialis) and clam (Ruditapes philippinarum) hemocytes after bacterial challenge. Four independent approaches (BestKeeper, GeNorm, NormFinder and DeltaCt ) were used to assess the suitable genes for stable expression. For these particular circumstances, the most stable gene in hemocytes was elongation factor 1 − α for mussels and α-tubulin for clams.

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Section: Discussionmentioning

confidence: 99%

“…Living Resour. 27, 147-152 (2014) but in mollusks (Martínez-Fernández et al 2010) and also in bivalves (Araya et al 2008;Cubero-Leon et al 2012;Dheilly et al 2011;Du et al 2013;Mauriz et al 2012;Siah et al 2012); because different species in different circumstances are able to modify their transcriptome to overcome the situation.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of reference genes ofMytilus galloprovincialisandRuditapes philippinaruminfected with three bacteria strains for gene expression analysis

Moreira

Pereiro

Costa

et al. 2014

Aquat. Living Resour.

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“…Assembled sequence which could be annotated as the same protein again with best hit was used for candidate reference gene searching. Finally, a total of 12 commonly used candidate reference genes [18,35,44-47], including ACT , GAPDH , Cytochrome C (CC), Cytochrome B (CB), elongation factor 1-β ( EF-1-β ), Ubiquitin ( UBQ ), TATA-box binding protein (TBP), 60S ribosomal protein L16 ( RPL16 ), DEAD-box RNA helicase ( HELI ), β-Tubulin ( TUB ), Cyclophilin A ( CYP ), and Histone H3.3 ( His3.3 ) were selected for expression stability analysis (Table 1). …”

Section: Methodsmentioning

confidence: 99%

“…For example, in scallops, the most frequently used reference gene in qRT-PCR is ACT , but without evaluation. By far, in the limited studies on reference gene selection for bivalve species, most of them focused on single tissues, such as haemocyte in parasite attacked flat oyster ( Ostrea edulis ) [18] and bacteria infected soft-shell clam ( Mya Arenaria ) [17], and gonad in mussel ( Mytilus edulis ) [35] and lion’s paw scallop ( Nodipectensubnodosus ) [36]. Although the expression stability of candidate reference genes was assessed in several tissues of Yesso scallop ( Patinopecten yessoensis ) with respect to starvation treatment, the reference genes were recommended for different single tissues, and those could be generally used among tissues were not evaluated or identified [37].…”

Section: Introductionmentioning

confidence: 99%

“…Meanwhile, gene expression variations during bivalve development, which play critical roles in the tremendous morphology changes across embryo/larva stages, have been extensively studied [29,38-40], while related reference gene evaluation was only reported in oyster [41]. On the other hand, the number of candidate reference genes evaluated in single bivalve species is mostly no more than six [18,35,36] mainly due to the previously limited availability of gene sequences, thus increased the chances of missing more stably expressed genes.…”

Section: Introductionmentioning

confidence: 99%

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Identification of Reference Genes for qRT-PCR Analysis in Yesso Scallop Patinopecten yessoensis

Feng

Xue

et al. 2013

PLoS ONE

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BackgroundBivalves comprise around 30,000 extant species and have received much attention for their importance in ecosystems, aquaculture and evolutionary studies. Despite the increasing application of real-time quantitative reverse transcription PCR (qRT-PCR) in gene expression studies on bivalve species, little research has been conducted on reference gene selection which is critical for reliable and accurate qRT-PCR analysis. For scallops, systematic evaluation of reference genes that can be used among tissues or embryo/larva stages is lacking, and β-actin (ACT) is most frequently used as qRT-PCR reference gene without validation.ResultsIn this study, 12 commonly used candidate reference genes were selected from the transcriptome data of Yesso scallop ( Patinopecten yessoensis ) for suitable qRT-PCR reference genes identification. The expression of these genes in 36 tissue samples and 15 embryo/larva samples under normal physiological conditions was examined by qRT-PCR, and their expression stabilities were evaluated using three statistic algorithms, geNorm, NormFinder, and comparative ∆Ct method. Similar results were obtained by the three approaches for the most and the least stably expressed genes. Final comprehensive ranking for the 12 genes combing the results from the three programs showed that, for different tissues, DEAD-box RNA helicase (HELI), ubiquitin (UBQ), and 60S ribosomal protein L16 (RPL16) were the optimal reference genes combination, while for different embryo/larva stages, gene set containing Cytochrome B (CB), Cytochrome C (CC), Histone H3.3 (His3.3), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recommended for qRT-PCR normalization. ACT was among the least stable genes for both adult tissues and embryos/larvae.ConclusionsThis work constitutes the first systematic analysis on reference genes selection for qRT-PCR normalization in scallop under normal conditions. The suitable reference genes we recommended will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species.

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Gonadal Atresia, Estrogen-Responsive, and Apoptosis-Specific mRNA Expression in Marine Mussels from the East China Coast: A Preliminary Study

Zhu

Chapman

et al. 2022

Bull Environ Contam Toxicol

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This preliminary survey analysed mussel atresia incidences, estrogen-responsive and apoptotic-specific molecular end points, and aqueous and gonadal levels of selected estrogens from the East China coast. Estrogen levels were low (e.g. < LOD-28.36 ng/L, < LOD-3.88 ng/g wet weight of tissue for BPA) relative to worldwide freshwater environments, but high oocyte follicle atresia incidences (up to 26.6%) occurred at selected sites. Expression of estrogen-responsive ER2 was significantly increased in males relative to females at sites with high atresia incidences in females. A second estrogen-responsive gene, V9, was significantly increased at two sites in April in females relative to males; the opposite was true for the remaining two sites. Apoptosis-specific genes (Bcl-2, fas) showed elevated expression in males relative to females at the site with the highest atresia incidence. These results provide coastal estrogen levels and the utility of several estrogen-specific molecular-level markers for marine mussels.

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Reference gene selection for qPCR in mussel, Mytilus edulis, during gametogenesis and exogenous estrogen exposure

Abstract: We demonstrate that the experimental results are highly dependent on the reference gene chosen and that statistically significant contrasting differences between sample groups are present or absent depending on the reference gene employed.

Cited by 62 publications

References 21 publications

Evaluation of reference genes ofMytilus galloprovincialisandRuditapes philippinaruminfected with three bacteria strains for gene expression analysis

Evaluation of reference genes ofMytilus galloprovincialisandRuditapes philippinaruminfected with three bacteria strains for gene expression analysis

Identification of Reference Genes for qRT-PCR Analysis in Yesso Scallop Patinopecten yessoensis

Gonadal Atresia, Estrogen-Responsive, and Apoptosis-Specific mRNA Expression in Marine Mussels from the East China Coast: A Preliminary Study

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