Reference genes to quantify gene expression during oogenesis in a teleost fish

Deloffre, Laurence; Andrade, André R.; Filipe, Alexandra; Canário, Adelino V.M.

doi:10.1016/j.gene.2012.06.047

Cited by 26 publications

(13 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Numerous studies have attempted to validate the expression stability of reference genes in the ovaries of vertebrates, including fish species. Moreover, actb, ctsd, ef1α, and cathepsin Z were regarded as the most stable gene at the maturation stages of the ovary in tilapia 5 . In the current study actb and ctsd were the two most suitable gene combinations for normalization in the ovary of turbot during development stages.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Reference gene validation for quantification of gene expression during ovarian development of turbot (Scophthalmus maximus)

Gao

Huang

et al. 2020

Sci Rep

View full text Add to dashboard Cite

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a powerful and sensitive method used in gene expression analysis. Suitable reference genes, which are stable under all experimental circumstances and tissues significantly improve the accuracy of qRT-PCR data. In this study, the stability of six genes, namely, 18S ribosomal RNA (18s), beta-actin (actb), elongation factor 1-alpha (ef1α), glyceraldehyde-3-phosphate-dehydrogenase (gapdh), cathepsin D (ctsd), and beta-2-microglobulin (b2m) were evaluated as potential references for qRT-PCR analysis. The genes were examined in the hypothalamus-pituitary-ovary-liver (HPOL) axis throughout turbot ovarian development via using the geNorm, NormFinder and BestKeeper algorithms. Results showed that the most stable reference genes were ef1α, actb, and ctsd in the hypothalamus, pituitary, ovary and liver, respectively. The best-suited gene combinations for normalization were 18s, ef1α, and ctsd in the hypothalamus; actb, ctsd, and 18s in the pituitary; actb, and ctsd in the ovary; gapdh and ctsd in the liver. Moreover, the expression profile of estrogen receptor α (erα) manifested no significant difference normalization to the aforementioned best-suited gene during turbot ovarian development. However, no single gene or pair of genes is suitable as an internal control and account for the amplification differences among the four tissues during ovarian development. In summary, these results provide a basic data for the optimal reference gene selection and obtain highly accurate normalization of qRT-PCR data in HPOL axis-related gene expression analysis during turbot ovarian development. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in gene expression analysis because of the advantages of its sensitivity and the accurate detection of mRNA at extremely low transcription levels 1. The successful application of qRT-PCR depends on accurate transcript normalization via the selection of suitable reference genes. The reference genes minimize errors attributed to the use of biological samples, such as experimental operations, and RNA and cDNA qualities 2. The ideal reference gene for qRT-PCR exhibits a stable expression level in all target tissues/cells and should be no affected by various experimental conditions or treatments. In general, the commonly used reference genes mainly include glyceraldehyde-3-phosphate-dehydrogenase (gapdh), 18S ribosomal RNA (18s), beta actin (actb), elongation factor 1-alpha (ef1α), cathepsin D (ctsd), and beta-2-microglobulin-like (b2m) in turbot 3,4 and other fish species 5-8. However, these classical reference genes vary at the transcription level in different tissues, at different developmental stages and experimental conditions or treatments 9-11. Inappropriate reference genes that serve as internal controls can affect the accuracy of qRT-PCR results and cause the significantly different conclusion 5,12,13. Actually, no single reference gene is universally applicable in ...

show abstract

Section: Discussionmentioning

confidence: 99%

“…BestKeeper evaluates the stability of candidate genes by calculating the standard deviation (SD) and correlation coefficient (r). In general, two or more software programs are used together during data analysis to identify discrepancies between the outputs of different algorithms 5,20,21 .…”

mentioning

confidence: 99%

Reference gene validation for quantification of gene expression during ovarian development of turbot (Scophthalmus maximus)

Gao

Huang

et al. 2020

Sci Rep

View full text Add to dashboard Cite

show abstract

“…There is massive variation in gene transcription within the gonads between the immature and mature stage, which also explains the variation in constitutive gene expression. In tilapia, transcription of the reference genes increases significantly during oogenesis, especially during periods of high vitellogenic protein production (Deloffre et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

SHORT-COMMUNICATION Validation of reference genes for real-time quantitative PCR in tambaqui (Colossoma macropomum)

et al. 2016

View full text Add to dashboard Cite

Tambaqui, Colossoma macropomum, is the main native freshwater fish in Brazilian aquaculture. Therefore, intensive research pressure has been applied to the species to support new technologies for tambaqui farming. Molecular biology represents a tool that can be used to investigate every field of applied biology, from fish physiology to the effects of climate change. Based on the importance of reference genes for the relative or absolute quantification of gene transcripts, we cloned and sequenced three candidate reference genes in tambaqui (18S ribossomal RNA - 18s, glyceraldehyde-3-phosphate dehydrogenase - gapdh, and actin beta - β-actin), and validated a set of primers for each gene for use in real-time quantitative PCR. The results were evaluated by RefFinder, which indicated that β-actin is the most suitable reference gene for tambaqui among those studied, followed by 18s.

show abstract

“…Thus, we opted for reporting non-normalized relative amounts for every target gene studied as proposed by Diaz de Cerio et al (2012) for C. labrosus. This strategy was successfully applied in European sea bass gonad (García-López et al, 2011a,b), Mozambique tilapia ovaries (Deloffre et al, 2012), Kuruma shrimp gonad (Sellars et al, 2007) and human seminoma cells (Neuvians et al, 2005). All C T values were compared with respect to the highest C T value recorded for each gene, which represents the individual showing the lowest mRNA transcription obtained for a specific gene (Díaz de Cerio et al, 2012).…”

Section: Quantification By Qpcrmentioning

confidence: 99%

Steroidogenesis and phase II conjugation during the gametogenesis of thicklip grey mullet (Chelon labrosus) from a population showing intersex condition

Sardi

Bizarro

Cajaraville

et al. 2015

General and Comparative Endocrinology

View full text Add to dashboard Cite

Reference genes to quantify gene expression during oogenesis in a teleost fish

Cited by 26 publications

References 43 publications

Reference gene validation for quantification of gene expression during ovarian development of turbot (Scophthalmus maximus)

Reference gene validation for quantification of gene expression during ovarian development of turbot (Scophthalmus maximus)

SHORT-COMMUNICATION Validation of reference genes for real-time quantitative PCR in tambaqui (Colossoma macropomum)

Steroidogenesis and phase II conjugation during the gametogenesis of thicklip grey mullet (Chelon labrosus) from a population showing intersex condition

Contact Info

Product

Resources

About