Abstract
Gene expression of reproductive system of the black tiger shrimp (Peneaus monodon) has been widely studied to address poor maturation problem in captivity. However, a systematic evaluation of reference genes in quantitative real-time PCR (qPCR) for P. monodon reproductive organs is lacking. In this study, the stability of four potential reference genes (18s rRNA, GAPDH, β-actin, and EF1-α) was examined in the reproductive tissues in various conditions using bioinformatic tools: NormFinder and geNorm. For NormFinder, EF1-α and GAPDH ranked first and second as the most stable genes in testis groups whereas GAPDH and EF1-α were for ovaries from wild-caught broodstock and domesticated groups. EF1-α and β-actin ranked first and second for the eyestalk ablated ovaries. For geNorm, EF1-α and GAPDH had the best stability in all testis and ovaries from domesticated groups whereas EF1-α and β-actin were the best for ovaries from wild-caught and eyestalk ablated groups. Moreover, the expression levels of two well-known reproductive genes, Dmc1 and Vitellogenin, were used to validate these reference genes. When normalized to EF1-α, the expected expression patterns were obtained in all cases. Therefore, this work suggests that EF1-α is more versatile as reference genes in qPCR analysis for reproductive system in P. monodon.
Highlights
Quantitative real-time polymerase chain reaction is a useful technique to measure gene expression levels due to its high sensitivity, accuracy, and reproducibility
For gene expression analysis of the reproductive system in the black tiger shrimp Peneaus monodon, samples differ between individuals, tissues, growth stages and developmental stages; yet no previous study has examined the most appropriate genes to be used as an internal control gene
Four commonly used housekeeping genes (18S rRNA, glyceraldehydes-3 phosphate dehygrogenase (GAPDH), b-actin, and elongation factor 1 alpha (EF-1a)) in quantitative real-time PCR (qPCR) gene expression analysis were validated for their suitability as a reference gene for reproductive organs of P. monodon
Summary
Quantitative real-time polymerase chain reaction (qPCR) is a useful technique to measure gene expression levels due to its high sensitivity, accuracy, and reproducibility. We validated four commonly used reference genes (18S rRNA, GAPDH, b-actin, and EF-1a) to be used as an internal control in qPCR analysis of reproductive samples with various conditions.
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