Selection of reference genes for quantitative RT-PCR studies on the gonad of the bivalve mollusc Pecten maximus L.

Mauriz, O.; Maneiro, Verónica; Pérez‐Parallé, M. Luz; Sánchez, José L.; Pazos, A.

doi:10.1016/j.aquaculture.2012.10.020

Cited by 23 publications

(18 citation statements)

References 27 publications

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“…It has also been associated with the regulation of cytoskeletal dynamics 26 and is expressed in a wide variety of tissues, including showing expression in tumour cells 27 28 . In a previous study by our group 29 , the transcription level of RPSA in the testis of P. americana was found to be 2.83-fold higher than in the ovary, in accord with a previous study in which the non-normalized gene expression level of RPSA in the testis was 6.3-fold higher than in the ovary 30 . Therefore, we speculate that RPSA may play a greater functional role in the testes than in the ovaries.…”

Section: Discussionsupporting

confidence: 88%

De novo Assembly and Characterization of the Testis Transcriptome and Development of EST-SSR Markers in the Cockroach Periplaneta americana

Chen

Liu

Jiang

2015

Sci Rep

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The cockroach Periplaneta americana is a notorious pest and threat to health worldwide, with a high reproductive ability. However, a limited amount of data is available on the developmental stage-specific transcriptomes of P. americana. To identify genes involved in developmental processes and to develop additional SSR markers in P. americana, we carried out de novo assembly of the P. americana transcriptome using Illumina sequencing. After removing low-quality sequences, we obtained 64,954,709 contigs, which were further assembled into 125,390 unigenes with an average length of 711 bp. Based on similarity searches against known proteins, we identified 48,300 unigenes based on a cut-off E-value of 10−5. The assembled sequences were annotated according to gene descriptions, gene ontology and clusters of orthologous groups. A total of 14,195 potential SSRs were identified, and 41 of 63 randomly chosen primer pairs successfully amplified the predicted SSR markers, seven of which were polymorphic in size in P. americana. Furthermore, the Spag6 gene was confirmed to be testes specific, and the fru and RPSA genes were related to the development of the testis. This is the special report of a P. americana transcriptome obtained using Illumina sequencing technology, and a large number of molecular markers were developed.

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Section: Discussionsupporting

confidence: 88%

De novo Assembly and Characterization of the Testis Transcriptome and Development of EST-SSR Markers in the Cockroach Periplaneta americana

Chen

Liu

Jiang

2015

Sci Rep

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“…This indicates that all seven genes showed some variation in expression, and that even all seven are not sufficient for normalization according to the criterion suggested by Vandesompele et al (2002) (V n/n + 1 < 0.15). In fact, similar to our results, high V n/n + 1 values (all over 0.15) were also observed in a study on the gonad of another bivalve species (Mauriz et al, 2012), although different candidate housekeeping genes were used. Obtaining relatively high V n/n + 1 values may be a common result in analyses using molluscan samples.…”

Section: Discussionsupporting

confidence: 77%

Assessment of housekeeping genes as internal references in quantitative expression analysis during early development of oyster

2016

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The early development of mollusks exhibits important characteristics from the developmental and evolutionary perspective. With the increasing number of genomewide studies, accurate analyses of quantitative gene expression during development are impeded by the lack of validated reference genes. To improve the situation, in this study, we analyzed the expression stability of seven candidate housekeeping genes during early development of the Pacific oyster Crassostrea gigas: actin, glyceraldehyde-3-phosphate dehydrogenase (gapdh), α subunit of elongation factor 1 (elf1α), adp-ribosylation factor 1 (arf1), heterogeneous nuclear ribonucleoprotein q, ubiquitin-conjugating enzyme e2d2 and ribosomal protein s18. We focused on 11 stages from oocyte to D-veliger, which include crucial developmental processes such as axis determination, gastrulation and shell formation. Gene expression stabilities were assessed with the three commonly used programs geNorm, NormFinder and BestKeeper. Although the results obtained with the three programs varied to some extent, in general, arf1, elf1α and gapdh were highly ranked and actin was poorly ranked. This analysis also indicated that multiple genes should be used for normalization, and we concluded that arf1-elf1α-gapdh should be used as internal references. The findings of this study will help researchers to obtain accurate results in future quantitative gene expression analysis of development in bivalve mollusks.

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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%

“…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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-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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Selection of reference genes for quantitative RT-PCR studies on the gonad of the bivalve mollusc Pecten maximus L.

Cited by 23 publications

References 27 publications

De novo Assembly and Characterization of the Testis Transcriptome and Development of EST-SSR Markers in the Cockroach Periplaneta americana

De novo Assembly and Characterization of the Testis Transcriptome and Development of EST-SSR Markers in the Cockroach Periplaneta americana

Assessment of housekeeping genes as internal references in quantitative expression analysis during early development of oyster

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

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