Abstract
The Nile tilapia (Oreochromis niloticus) is one of the most important cultured fish worldwide, but tilapia culture is largely affected by low temperatures. Recent studies suggest that microRNAs (miRNAs) regulate cold tolerance traits in fish. In general, qPCR-based methods are the simplest and most accurate forms of miRNA quantification. However, qPCR data heavily depends on appropriate normalization. Therefore, the aim of the present study is to determine whether the expression of previously tested, stably expressed miRNAs are affected by acute cold stress in Nile tilapia. For this purpose, one small nuclear RNA (U6) and six candidate reference miRNAs (miR-23a, miR-25–3, Let-7a, miR-103, miR-99–5, and miR-455) were evaluated in four tissues (blood, brain, liver, and gills) under two experimental conditions (acute cold stress and control) in O. niloticus. The stability of the expression of each candidate reference miRNA was analyzed by four independent methods (the delta Ct method, geNorm, NormFinder, and BestKeeper). Further, consensual comprehensive ranking of stability was built with RefFinder. Overall, miR-103 was the most stable reference miRNA in this study, and miR-103 and Let-7a were the best combination of reference targets. Equally important, Let-7a, miR-23a, and miR-25–3 remained consistently stable across different tissues and experimental groups. Considering all variables, U6, miR-99–5, and miR-455 were the least stable candidates under acute cold stress. Most important, suitable reference miRNAs were validated in O. niloticus, facilitating further accurate miRNA quantification in this species.
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Data that support the findings of this study are available from the corresponding author upon reasonable request.
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References
Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64(15):5245–5250. https://doi.org/10.1158/0008-5472.can-04-0496
Andreassen R, Rangnes F, Sivertsen M, Chiang M, Tran M, Worren MM (2016) Discovery of miRNAs and their corresponding miRNA genes in Atlantic cod (Gadus morhua): use of stable miRNAs as reference genes reveals subgroups of miRNAs that are highly expressed in particular organs. PLOS ONE 11(4):e0153324. https://doi.org/10.1371/journal.pone.0153324
Benz F, Roderburg C, Vargas Cardenas D, Vucur M, Gautheron J, Koch A, Zimmermann H, Janssen J, Nieuwenhuijsen L, Luedde M, Frey N, Tacke F, Trautwein C, Luedde T (2013) U6 is unsuitable for normalization of serum miRNA levels in patients with sepsis or liver fibrosis. Exp Mol Med 45(9):e42–e42. https://doi.org/10.1038/emm.2013.81
Bizuayehu TT, Babiak I (2014) MicroRNA in Teleost fish. Genome Biol Evol 6(8):1911–1937. https://doi.org/10.1093/gbe/evu151
Blödorn EB, Domingues WB, Nunes LS, Komninou ER, Pinhal D, Campos VF (2021) MicroRNA roles and their potential use as selection tool to cold tolerance of domesticated teleostean species: A systematic review. Aquaculture 540:736747. https://doi.org/10.1016/j.aquaculture.2021.736747
Cardona E, Milhade L, Pourtau A, Panserat S, Terrier F, Lanuque A, Roy J, Marandel L, Bobe J, Skiba-Cassy S (2022) Tissue origin of circulating microRNAs and their response to nutritional and environmental stress in rainbow trout (Oncorhynchus mykiss). Sci Total Environ 853(1):158584. https://doi.org/10.1016/j.scitotenv.2022.158584
Cardona E, Guyomar C, Desvignes T, Montfort J, Guendouz S, Postlethwait JH, Skiba-Cassy S, Bobe J (2021) Circulating miRNA repertoire as a biomarker of metabolic and reproductive states in rainbow trout. BMC Biol 19(1). https://doi.org/10.1186/s12915-021-01163-5
Chen C (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179–e179. https://doi.org/10.1093/nar/gni178
Davoren PA, McNeill RE, Lowery AJ, Kerin MJ, Miller N (2008) Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer. BMC Mol Biol 9(1):76. https://doi.org/10.1186/1471-2199-9-76
FAO, Food and Agriculture Organization of the United Nations (2018) The state of world fisheries and aquaculture: meeting the sustainable development goals. Italy, Rome
Forero DA, González-Giraldo Y, Castro-Vega LJ, Barreto GE (2019) qPCR-based methods for expression analysis of miRNAs. Biotechniques 67(4):192–199. https://doi.org/10.2144/btn-2019-0065
Fu Y-S, Shi Z-Y, Wang G-Y, Li W-J, Zhang J-L, Jia L (2012) Expression and regulation of miR-1, -133a, -206a, and MRFs by thyroid hormone during larval development in Paralichthys olivaceus. Comp Biochem Physiol Part B Biochem Mol Biol 161(3):226–232. https://doi.org/10.1016/j.cbpb.2011.11.009
Gu Y, Li M, Zhang K, Chen L, Jiang A, Wang J, Lv X, Li X (2011) Identification of suitable endogenous control microRNA genes in normal pig tissues. Anim Sci J 82(6):722–728. https://doi.org/10.1111/j.1740-0929.2011.00908.x
Herkenhoff ME, Oliveira AC, Nachtigall PG, Costa JM, Campos VF, Hilsdorf AWS, Pinhal D (2018) Fishing into the microRNA transcriptome. Front Genet 9(88). https://doi.org/10.3389/fgene.2018.00088
Huang CW, Li YH, Hu SY, Chi JR, Lin GH, Lin CC, Gong HY, Chen JY, Chen RH, Chang SJ, Liu FG, Wu JL (2012) Differential expression patterns of growth-related microRNAs in the skeletal muscle of Nile tilapia (Oreochromis niloticus). J Anim Sci 90(12):4266–4279. https://doi.org/10.2527/jas.2012-5142
Hung I-C, Hsiao Y-C, Sun HS, Chen T-M, Lee S-J (2016). MicroRNAs regulate gene plasticity during cold shock in zebrafish larvae. BMC Genomics 17(1). https://doi.org/10.1186/s12864-016-3239-4
Ikert H, Lynch MDJ, Doxey AC, Giesy JP, Servos MR, Katzenback BA, Craig PM (2021) High throughput sequencing of microRNA in rainbow trout plasma, mucus, and surrounding water following acute stress. Front Physiol 11(588313). https://doi.org/10.3389/fphys.2020.588313
Johansen I, Andreassen R (2014) Validation of miRNA genes suitable as reference genes in qPCR analyses of miRNA gene expression in Atlantic salmon (Salmo salar). BMC Res Notes 8(1):945. https://doi.org/10.1186/1756-0500-7-945
Lardizábal MN, Nocito AL, Daniele SM, Ornella LA, Palatnik JF, Veggi LM (2012) Reference genes for real-time PCR quantification of microRNAs and messenger RNAs in rat models of hepatotoxicity. PLoS ONE 7(5):e36323. https://doi.org/10.1371/journal.pone.0036323
Lau K, Lai KP, Bao JYJ, Zhang N, Tse A, Tong A, Li JW, Lok S, Kong RYC, Lui WY, Wong A, Wu RSS (2014) Identification and expression profiling of microRNAs in the brain, liver and gonads of marine medaka (Oryzias melastigma) and in response to hypoxia. PLOS ONE 9(10):e110698. https://doi.org/10.1371/journal.pone.0110698
Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433(7027):769–773. https://doi.org/10.1038/nature03315
Liu J, Jia E, Shi H, Li X, Jiang G, Chi C, Liu W, Zhang D (2019) Selection of reference genes for miRNA quantitative PCR and its application in miR-34a/Sirtuin-1 mediated energy metabolism in Megalobrama amblycephala. Fish Physiol Biochem 45(5):1663–1681. https://doi.org/10.1007/s10695-019-00658-x
Liu S, Song H, Liu Z, Lu W, Zhang Q, Cheng J (2022) Selection of references for microRNA quantification in Japanese flounder (Paralichthys olivaceus) normal tissues and Edwardsiella tarda-infected livers. Genes 13(2):175. https://doi.org/10.3390/genes13020175
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Lou G, Ma N, Xu Y, Jiang L, Yang J, Wang C, Jiao Y, Gao X (2015) Differential distribution of U6 (RNU6-1) expression in human carcinoma tissues demonstrates the requirement for caution in the internal control gene selection for microRNA quantification. Int J Mol Med 36(5):1400–1408. https://doi.org/10.3892/ijmm.2015.2338
Ma XY, Qiang J, He J, Gabriel NN, Xu P (2015) Changes in the physiological parameters, fatty acid metabolism, and SCD activity and expression in juvenile GIFT tilapia (Oreochromis niloticus) reared at three different temperatures. Fish Physiol Biochem 41(4):937–950. https://doi.org/10.1007/s10695-015-0059-4
Nachtigall PG, Dias MC, Carvalho RF, Martins C, Pinhal D (2015) MicroRNA-499 expression distinctively correlates to target genes sox6 and rod1 profiles to resolve the skeletal muscle phenotype in Nile tilapia. PLOS ONE 10(3):e0119804. https://doi.org/10.1371/journal.pone.0119804
Nie M, Tan X, Lu Y, Wu Z, Li J, Xu D, Zhang P, You F (2019) Network of microRNA-transcriptional factor-mRNA in cold response of turbot Scophthalmus maximus. Fish Physiol Biochem 45(2):583–597. https://doi.org/10.1007/s10695-019-00611-y
Nitzan T, Kokou F, Doron-Faigenboim A, Slosman T, Biran J, Mizrahi I, Zak T, Benet A, Cnaani A (2019) Transcriptome analysis reveals common and differential response to low temperature exposure between tolerant and sensitive blue tilapia (Oreochromis aureus). Front Genet 10:100. https://doi.org/10.3389/fgene.2019.00100
Nobrega RO, Batista RO, Corrêa CF, Mattioni B, Filer K, Pettigrew JE, Fracalossi DM (2019) Dietary supplementation of Aurantiochytrium sp. meal, a docosahexaenoic-acid source, promotes growth of Nile tilapia at a suboptimal low temperature. Aquaculture 507:500–509. https://doi.org/10.1016/j.aquaculture.2019.04.030
Nobrega RO, Banze JF, Batista RO, Fracalossi DM (2020) Improving winter production of Nile tilapia: What can be done? Aquac Rep 18:100453. https://doi.org/10.1016/j.aqrep.2020.100453
O’Brien J, Hayder H, Zayed Y, Peng C (2018) Overview of microRNA biogenesis, mechanisms of actions, and circulation. Front Endocrinol 9(402). https://doi.org/10.3389/fendo.2018.00402
Peltier HJ, Latham GJ (2008) Normalization of microRNA expression levels in quantitative RT-PCR assays: Identification of suitable reference RNA targets in normal and cancerous human solid tissues. RNA 14(5):844–852. https://doi.org/10.1261/rna.939908
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations. Biotech Lett 26(6):509–515. https://doi.org/10.1023/b:bile.0000019559.84305.47
Qiang J, Cui YT, Tao FY, Bao WJ, He J, Li XH, Xu P, Sun LY (2018). Physiological response and microRNA expression profiles in head kidney of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) exposed to acute cold stress. Sci Rep 8(1). https://doi.org/10.1038/s41598-017-18512-6
Rassier GT, Silveira TLR, Remião MH, Daneluz LO, Martins AWS, Dellagostin EN, Ortiz HG, Domingues WB, Komninou ER, Kütter MT, Marins LFF, Campos VF (2020) Evaluation of qPCR reference genes in GH-overexpressing transgenic zebrafish (Danio rerio). Sci Rep 10(1). https://doi.org/10.1038/s41598-020-69423-y
Sadoul B, Geffroy B (2019) Measuring cortisol, the major stress hormone in fishes. J Fish Biol 94(4):540–555. https://doi.org/10.1111/jfb.13904
Silveira TLR, Domingues WB, Remião MH, Santos L, Barreto B, Lessa IM, Varela Junior AS, Martins Pires D, Corcini C, Collares T, Seixas FK, Robaldo RB, Campos VF (2018) Evaluation of reference genes to analyze gene expression in silverside Odontesthes humensis under different environmental conditions. Front Genet 9. https://doi.org/10.3389/fgene.2018.00075
Silver N, Best S, Jiang J, Thein S (2006) Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 7(1):33. https://doi.org/10.1186/1471-2199-7-33
Sun J, Zhao L, Wu H, Lian W, Cui C, Du Z, Luo W, Li M, Yang S (2018) Analysis of miRNA-seq in the liver of common carp (Cyprinus carpio L.) in response to different environmental temperatures. Funct Integr Genomics 19(2):265–280. https://doi.org/10.1007/s10142-018-0643-7
Sun JL, Liu Q, Zhao L, Cui C, Wu H, Liao L, Tang G, Yang S, Yang S (2019) Potential regulation by miRNAs on glucose metabolism in liver of common carp (Cyprinus carpio) at different temperatures. Comp Biochem Physiol Part D Genomics Proteomics 32:100628. https://doi.org/10.1016/j.cbd.2019.100628
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):RESEARCH0034. https://doi.org/10.1186/gb-2002-3-7-research0034
Velmurugan BK, Chan C, Weng C (2019) Innate-immune responses of tilapia (Oreochromis mossambicus) exposure to acute cold stress. J Cell Physiol 234(9):16125–16135. https://doi.org/10.1002/jcp.28270
Wang F, Yang Q, Zhao W-J, Du Q-Y, Chang Z-J (2019) Selection of suitable candidate genes for miRNA expression normalization in Yellow River Carp (Cyprinus carpio. var). Sci Rep 9(1). https://doi.org/10.1038/s41598-019-44982-x
Witwer KW, Halushka MK (2016) Toward the promise of microRNAs – Enhancing reproducibility and rigor in microRNA research. RNA Biol 13(11):1103–1116. https://doi.org/10.1080/15476286.2016.1236172
Wu SM, Liu J-H, Shu L-H, Chen CH (2015) Anti-oxidative responses of zebrafish (Danio rerio) gill, liver and brain tissues upon acute cold shock. Comp Biochem Physiol Part A Mol Integr Physiol 187:202–213. https://doi.org/10.1016/j.cbpa.2015.05.016
Xie F, Xiao P, Chen D, Xu L, Zhang B (2012) miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant Mol Biol 80(1):75–84. https://doi.org/10.1007/s11103-012-9885-2
Xu X-Y, Shen Y-B, Fu J-J, Lu L-Q, Li J-L (2014) Determination of reference microRNAs for relative quantification in grass carp (Ctenopharyngodon idella). Fish Shellfish Immun 36(2):374–382. https://doi.org/10.1016/j.fsi.2013.12.007
Yan B, Zhao L, Guo J, Zhao J (2012) miR-206 regulates the growth of the teleost tilapia (Oreochromis niloticus) through the modulation of IGF-1 gene expression. J Exp Biol 216(7):1265–1269. https://doi.org/10.1242/jeb.079590
Yang C, Jiang M, Wen H, Tian J, Liu W, Wu F, Gou G (2015) Analysis of differential gene expression under low-temperature stress in Nile tilapia (Oreochromis niloticus) using digital gene expression. Gene 564(2):134–140. https://doi.org/10.1016/j.gene.2015.01.038
Yang R, Dai Z, Chen S, Chen L (2011) MicroRNA-mediated gene regulation plays a minor role in the transcriptomic plasticity of cold-acclimated Zebrafish brain tissue. BMC Genomics 12(1). https://doi.org/10.1186/1471-2164-12-605
Yilmaz S, Ergün S, Çelik EŞ, Banni M, Ahmadifar E, Dawood MAO (2021) The impact of acute cold water stress on blood parameters, mortality rate and stress-related genes in Oreochromis niloticus, Oreochromis mossambicus and their hybrids. J Therm Biol 100:103049. https://doi.org/10.1016/j.jtherbio.2021.103049
Zavala E, Reyes D, Deerenberg R, Vidal R (2017) Selection of reference genes for microRNA analysis associated to early stress response to handling and confinement in Salmo salar. Sci Rep 7(1). https://doi.org/10.1038/s41598-017-01970-3
Zhu X, Li Y-L, Chen D-X, Wu P, Yi T, Chen T, Zhang J-S, Chu W-Y (2015) Selection of reference genes for microRNA quantitative expression analysis in Chinese perch, Siniperca Chuatsi. Int J Mol Sci 16(12):8310–8323. https://doi.org/10.3390/ijms16048310
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This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/MCTI/CT-BIOTEC Nº 31/2022 #440636/2022-1), Fundação de Amparo à pesquisa do Estado do Rio Grande do Sul (FAPERGS#22/2551–0001645-6) and was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES) Finance Code 001 and AUXPE #2537/2018. VFC is also individually supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico.
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EBB was responsible for the conceptualization of this study, executed most experimentation steps, conducted all data analysis, and wrote the first draft of the manuscript. WBD contributed with the conceptualization of this study, reviewed and edited the manuscript. AWSM contributed with most of the experimentation steps, including sample collection, RNA extraction, cDNA synthesis, and qPCR reactions. END contributed with most of the experimentation steps, including sample collection, RNA extraction, cDNA synthesis, and qPCR reactions. ERK contributed with animal experimentation and sample collection. JLG contributed with the cortisol analysis. RAV contributed with the cortisol analysis. GLC provided appropriate conditions for fish experimentation at the "Laboratório de Piscicultura da Barragem do Chasqueiro". TLRS contributed with qPCR data analysis for the determination of the best reference targets, reviewed and edited the manuscript. MHR contributed with qPCR data analysis for the determination of the best reference targets, reviewed and edited the manuscript. VFC was responsible for supervision, project administration, funding acquisition, review and editing the manuscript. All authors read and approved the final version of the manuscript.
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Blödorn, E.B., Domingues, W.B., Martins, A.W.S. et al. MicroRNA qPCR normalization in Nile tilapia (Oreochromis niloticus): Effects of acute cold stress on potential reference targets. Fish Physiol Biochem 49, 409–423 (2023). https://doi.org/10.1007/s10695-023-01190-9
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DOI: https://doi.org/10.1007/s10695-023-01190-9