Abstract
The present study was aimed at screening suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) in tiger puffer (Takifugu rubripes), an important aquaculture species in Asia and also a good model species for lipid research. Specifically, this reference gene screening was targeted at standardization of gene expression in different tissues (liver, muscle, brain, intestine, heart, eye, skin, and spleen) or under different nutritional conditions (starvation and different dietary lipid levels). Eight candidate reference genes (ribosomal protein L19 and L13 (RPL19 and RPL13), elongation factor-1 alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase1 (HPRT1), beta-2-Microglobulin (B2M), 18S ribosomal RNA (18SrRNA), and beta actin (ACTB)) were evaluated with four algorithms (geNorm, NormFinder, BestKeeper, and comparative ΔCt method). The results showed that different algorithms generated inconsistent results. Based on these findings, RPL19, EF1α, 18SrRNA, and RPL13 were relatively stable in different tissues of tiger puffer. During starvation conditions, ACTB/RPL19 was the best reference gene combination. Under different dietary lipid levels, ACTB/RPL13 was the most suitable reference gene combination. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in tiger puffer.
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Abbreviations
- RPL19 :
-
Ribosomal protein L19
- RPL13 :
-
Ribosomal protein L13
- EF1 α :
-
Elongation factor-1 alpha
- GAPDH :
-
Glyceraldehyde-3-phosphate dehydrogenase
- HPRT1 :
-
Hypoxanthine guanine phosphoribosyl transferase1
- B2M :
-
Beta-2-Microglobulin
- 18SrRNA :
-
18S ribosomal RNA
- ACTB :
-
Beta actin
- qRT-PCR:
-
Quantitative real-time reverse-transcriptase polymerase chain reaction
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Funding
This work was supported by the National Key R&D Program of China (2018YFD0900400), China Agriculture Research System (CARS-47-G15), National Natural Science Foundation of China (31772862), and Central Public-interest Scientific Institution Basal Research Fund, CAFS (2020TD48).
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ZL conducted the feeding trial, did the PCR studies, and wrote the manuscript; ZS and QB participated in the feeding trial; QG and HX designed the study; BS and YW participated in the sampling and analysis; ML and HX reviewed the manuscript.
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All sampling protocols, as well as fish rearing practices, were reviewed and approved by the Animal Care and Use Committee of Yellow Sea Fisheries Research Institute.
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Highlights
1. This was the first study to screen suitable reference genes for tiger puffer among different tissues and under different nutritional conditions.
2. RPL19, EF1α, 18SrRNA, and RPL13 were suitable reference genes, while the HPRT1 and GAPDH appeared unsuitable as reference genes for qRT-PCR studies concerning different tissues of tiger puffer.
3. Under feed deprivation, ACTB/RPL19 was the most stable reference gene combination.
4. In response to different dietary lipid levels, ACTB/RPL13 was a suitable reference gene combination.
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Liao, Z., Sun, Z., Bi, Q. et al. Screening of reference genes in tiger puffer (Takifugu rubripes) across tissues and under different nutritional conditions. Fish Physiol Biochem 47, 1739–1758 (2021). https://doi.org/10.1007/s10695-021-01012-w
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DOI: https://doi.org/10.1007/s10695-021-01012-w