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

Abstract

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 ...

Selection and Verification of Reference Genes for Gene Expression Studies in Different Cell Lines of Golden Pompano (Trachinotus ovatus)

“…As previously reported, the rankings of the most stable reference genes assessed via different software manifested similarity to a certain extent, but slight discrepancies due to adopting of various algorithms also occurred [62]. For example, in the pituitary of turbot, the results of geNorm demonstrated that actb and ctsd were the most suitable reference genes, and the optimal candidates assessed by Normfinder were actb and b2m, whereas in accordance BestKeeper analysis, 18S was the most appropriate reference gene [67]. Consistently, in this study, the three software all proposed 18S as the most suitable candidate in GPS cells under normal physiological conditions; regarding GPHK cells, in the light of results generated by BestKeeper or geNorm, the optimal reference genes were RPL13 and RPL13/18S, respectively, whereas UBCE was evaluated as the most stable reference gene according to NormFinder.…”

Selection and validation of reference genes by RT-qPCR for murine cementoblasts in mechanical loading experiments simulating orthodontic forces in vitro

“…RT-qPCR is a commonly used method to study changes in gene expression due to its capacity of relative quantification of gene expression. It is always necessary to find at least one or better multiple individually tested reference genes for each cell type through experimental condition considering the error susceptibility and sensitivity of this method 35 .…”

Evaluation of housekeeping genes as references for quantitative real‐time PCR analysis of European eel, Anguilla anguilla

“…ACTB was reported to be used for qPCR profiling of tissue‐specific gene expression in half‐smooth tongue sole (Li et al ., 2010) and blotched snakehead ( C. maculata ) (Mao et al ., 2017). GAPDH is considered a favourable reference gene in common carp ( Cyprinus carpio ) (Shi et al ., 2016), turbot ( Scophthalmus maximus ) (Gao et al ., 2020) and embryonic zebrafish ( D. rerio ) (Liu et al ., 2018). 18 S was also rated as the most stable reference gene in many fish tissues (Wang et al ., 2018; Liu et al ., 2014; Small et al ., 2008).…”