Identification and evaluation of reference genes for expression studies by RT-qPCR during embryonic development of the emerging model organism, Macrobrachium olfersii

Jaramillo, Michael L.; Ammar, Dib; Quispe, Ruth L.; Guzmán, Frank; Margis, Rogério; Nazari, Evelise Maria; Müller, Yara Maria Rauh

doi:10.1016/j.gene.2016.11.001

Cited by 28 publications

(26 citation statements)

References 51 publications

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“…Reference genes commonly used in gene expression studies during larval development of L. vannamei include β‐actin , rpS6 (40S ribosomal protein S6), and 18S rRNA (Lage et al, 2017; Quispe et al, 2016; Wei et al, 2014). Few studies regarding validation of reference genes have been performed in different crustacean species; for example, in Homarus americanus four candidate reference genes ( 18S rRNA, GAPDH, β ‐Actin , and EF1α ) were evaluated for gene expression quantification in reproductive organs, suggesting EF1α as the most stable (Hines, Clark, & Greenwood, 2014); in Macrobrachium olfersii six reference genes ( β‐Actin , GAPDH , EF1α , RpL8 , RpS6 , AK ) were analysed in embryos and in adult tissues (cerebral ganglia, muscle and hepatopancreas), suggesting RpL8 and RpS6 as the most stable genes (Jaramillo et al, 2017); in P. stylirostris the expression stability of 18S rRNA, GAPDH, β‐Actin and EF1α was measured in healthy and white spot syndrome virus (WSSV)‐infected shrimp, reporting GAPDH as the most stable gene followed by EF1 (Dhar, Bowers, Licon, Veazey, & Read, 2009); and in Procambarus clarkii eight candidate reference genes ( GAPDH , β‐Actin , EF1α UB‐Ubiquitin , β‐Tubulin , TBP , EIF , 18S rRNA ) were evaluated in different tissues, reporting EIF and 18S rRNA as the most stable genes, although TBP and EIF were more stable in the ovary (Jiang et al, 2015). These results indicate that there are no universal reference genes for all cases, and each experimental model needs validation.…”

Section: Discussionmentioning

confidence: 99%

“…Metabolic enzymes such as GAPDH and PK participate in glycolysis, although they perform other roles (Jung et al, 2012). GAPDH has been reported to be a good reference gene in experimental assays using adult shrimp (Dhar et al, 2009; Valenzuela‐Castillo et al, 2017), however, it was not the best option for measuring gene expression in embryonic stages of M. olfersii (Jaramillo et al, 2017). This could be related to the metabolic changes occurring in metamorphic stages; for instance, during the inter‐molting stage, glycolytic pathways are active, however, these pathways stop when animals enter to the pre‐molting stage, depleting the GAPDH protein requirement (Gao et al, 2017 ) .…”

Section: Discussionmentioning

confidence: 99%

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Selection of reference genes for the study of relative gene expression during development of Litopenaeus vannamei larvae

et al. 2020

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Pacific whiteleg shrimp (Litopenaeus vannamei) production is one of the most economically important aquaculture industries around the world. Gene expression studies during larval development are a key tool to elucidate biological aspects during metamorphosis and the effects of pathological conditions; the adequate quantification of gene expression can provide molecular markers and basic knowledge to optimize culture conditions, including the development of strategies for the prevention and control of diseases. In this context, the selection and validation of reference genes is a critical process to avoid bias due to inaccurate measurements and the consequent misinterpretation of biological processes. In this study, nine candidate reference genes (Clathrin, Cyt-c, SubF0, EF1α, β-Actin, GAPDH, TBP, AK and PK) were selected to test their expression stability during larval development of L. vannamei using geNorm, NormFinder, BestKeeper and the integrated tool RefFinder algorithms.Based on our analysis the most stable gene was SubF0, followed by GAPDH and EF1α.Relative expression of developmental genes (Twist, Mef-2 and Ubx) was quantified using the three most stable reference genes (individually and combined), taking special attention to the expected expression and biological function of these genes. The expression of Mef-2 was the most sensitive to the reference gene. However, the combination of the three most stable reference genes provided consistent results according to the expected expression patterns of developmental genes. K E Y W O R D Sgene expression, larval stages, Litopenaeus vannamei, reference genes, reverse transcription quantitative real-time polymerase chain reaction

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Selection of reference genes for the study of relative gene expression during development of Litopenaeus vannamei larvae

et al. 2020

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“…EIF was stable in different situations, especially in WSSV infection and in different ovarian and embryo stages, while RPL18 was the most stable reference gene in the adult tissues, and β-act was the best reference gene under hypoxia stress and RNAi. The five tools (GeNorm, NormFinder, BestKeeper, the comparative ∆C t method, and RefFinder) used in this research for analyzing the stability rankings of the reference gene were widely used in crustaceans [12,13,14,15,16], but the reference genes have rarely been screened for conditions of hypoxia and RNAi. The current reference genes, identified under different conditions in M. nipponense , will thus provide a useful references for qPCR experiments in other crustaceans.…”

Section: Discussionmentioning

confidence: 99%

“…In Peneaus monodon , the appropriate reference gene for the reproductive gene expression profile was identified [13]. Furthermore, the reference genes were screened before starting a quantitative study of heat-shock responses in Palaemonetes varians [14], while studies in Macrobrachium rosenbergii and Macrobrachium olfersii showed that reference genes were not universal, and the most appropriate reference gene depended on the specific conditions [15,16]. …”

Section: Introductionmentioning

confidence: 99%

Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR in Macrobrachium Nipponense

Qiao

et al. 2018

IJMS

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Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), β-act (β-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and β-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.

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“…PCR cycling conditions were set as follows: 2 min at 95 • C, 40 cycles of 5 s at 95 • C, and 10 s at 60 • C. Three biological replicates were used for all experiments and for each biological replicate, and three technical replicates were employed [49]. The relative expression of each predicted gene was calculated by the comparative 2 −∆∆ct method [50] with RpS6 used as the housekeeping gene [51].…”

Section: Reverse Transcription Quantitative Polymerase Chain Reactionmentioning

confidence: 99%

Comparative Transcriptome Analyses during the Vegetative Cell Cycle in the Mono-Cellular Organism Pseudokeronopsis erythrina (Alveolata, Ciliophora)

Shen

Gentekaki

et al. 2020

Microorganisms

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Studies focusing on molecular mechanisms of cell cycles have been lagging in unicellular eukaryotes compared to other groups. Ciliates, a group of unicellular eukaryotes, have complex cell division cycles characterized by multiple events. During their vegetative cell cycle, ciliates undergo macronuclear amitosis, micronuclear mitosis, stomatogenesis and somatic cortex morphogenesis, and cytokinesis. Herein, we used the hypotrich ciliate Pseudokeronopsis erythrina, whose morphogenesis has been well studied, to examine molecular mechanisms of ciliate vegetative cell cycles. Single-cell transcriptomes of the growth (G) and cell division (D) stages were compared. The results showed that (i) More than 2051 significantly differentially expressed genes (DEGs) were detected, among which 1545 were up-regulated, while 256 were down-regulated at the D stage. Of these, 11 randomly picked DEGs were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR); (ii) Enriched DEGs during the D stage of the vegetative cell cycle of P. erythrina were involved in development, cortex modifications, and several organelle-related biological processes, showing correspondence of molecular evidence to morphogenetic changes for the first time; (iii) Several individual components of molecular mechanisms of ciliate vegetative division, the sexual cell cycle and cellular regeneration overlap; and (iv) The P. erythrina cell cycle and division have the same essential components as other eukaryotes, including cyclin-dependent kinases (CDKs), cyclins, and genes closely related to cell proliferation, indicating the conserved nature of this biological process. Further studies are needed focusing on detailed inventory and gene interactions that regulate specific ciliated cell-phase events.

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Identification and evaluation of reference genes for expression studies by RT-qPCR during embryonic development of the emerging model organism, Macrobrachium olfersii

Cited by 28 publications

References 51 publications

Selection of reference genes for the study of relative gene expression during development of Litopenaeus vannamei larvae

Selection of reference genes for the study of relative gene expression during development of Litopenaeus vannamei larvae

Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR in Macrobrachium Nipponense

Comparative Transcriptome Analyses during the Vegetative Cell Cycle in the Mono-Cellular Organism Pseudokeronopsis erythrina (Alveolata, Ciliophora)

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