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

Understanding the molecular events involved in the acquisition of competence during oogenesis is a key step to determine the secret of ‘high quality’ eggs for aquaculture. Quantitative real time polymerase chain reaction (qPCR) is the technique of election to determine changes in transcript abundanc...

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Bibliographic Details
Published in:Gene 2012-09, Vol.506 (1), p.69-75
Main Authors: Deloffre, Laurence A.M., Andrade, André, Filipe, Alexandra I., Canario, Adelino V.M.
Format: Article
Language:English
Subjects:
actin
Actins - genetics
Animals
aquaculture
Base Sequence
binding proteins
bone morphogenetic proteins
cathepsin D
Cathepsin D - genetics
cathepsin X
Cathepsin Z - genetics
DNA Primers - genetics
eggs
Female
fish
Fish Proteins - genetics
Gene Expression
genes
Genetic Markers
messenger RNA
Normalization
Oocyte maturation stage
oocytes
Oogenesis - genetics
Oreochromis mossambicus
Peptide Elongation Factor 1 - genetics
qPCR
quantitative polymerase chain reaction
Real-Time Polymerase Chain Reaction
reverse transcription
TATA-Box Binding Protein - genetics
Tilapia - genetics
Tilapia - physiology
tubulin
Tubulin - genetics
vitellogenesis
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Summary:Understanding the molecular events involved in the acquisition of competence during oogenesis is a key step to determine the secret of ‘high quality’ eggs for aquaculture. Quantitative real time polymerase chain reaction (qPCR) is the technique of election to determine changes in transcript abundance in such studies, but choosing reference genes for normalization, in particular during oogenesis, remains a challenge. In the present study, transcription of 6 functionally distinct genes, β actin (ACTB), cathepsin D (CTSD), cathepsin Z (CTSZ), elongation factor 1 α (EEF1A), TATA binding protein (TBP) and tubulin A (TUBA1A) was assessed as normalizers of bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) gene expression in mRNA from Mozambique tilapia oocytes during oogenesis. Reverse transcription was equally efficient and varies little in all samples. Most of the genes considered for reference were stable during early stages of oogenesis but variations were observed during vitellogenesis. A single gene and up to 3 genes were shown to be insufficient for reliable normalization throughout the whole oogenesis. The combination of the genes ACTB, CTSD, EEF1A and CTSZ as reference was found to minimize variation and has the most stable expression pattern between maturation stages. ► Candidate reference genes were evaluated for expression stability during oogenesis. ► A 4-gene combination, ACTB, CTSD, EEF1A, and CTSZ, was assessed as normalizer. ► BAMBI expression in ovarian follicles decreased during vitellogenesis.
ISSN:0378-1119
1879-0038
DOI:10.1016/j.gene.2012.06.047