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Evaluation of reference genes for RT-qPCR study in abalone Haliotis discus hannai during heavy metal overload stress

  • Lee, Sang Yoon (Department of Marine Bio-Materials & Aquaculture, Pukyong National University) ;
  • Nam, Yoon Kwon (Department of Marine Bio-Materials & Aquaculture, Pukyong National University)
  • Received : 2016.02.27
  • Accepted : 2016.06.11
  • Published : 2016.06.30

Abstract

Background: The evaluation of suitable reference genes as normalization controls is a prerequisite requirement for launching quantitative reverse transcription-PCR (RT-qPCR)-based expression study. In order to select the stable reference genes in abalone Haliotis discus hannai tissues (gill and hepatopancreas) under heavy metal exposure conditions (Cu, Zn, and Cd), 12 potential candidate housekeeping genes were subjected to expression stability based on the comprehensive ranking while integrating four different statistical algorithms (geNorm, NormFinder, BestKeeper, and ${\Delta}CT$ method). Results: Expression stability in the gill subset was determined as RPL7 > RPL8 > ACTB > RPL3 > PPIB > RPL7A > EF1A > RPL4 > GAPDH > RPL5 > UBE2 > B-TU. On the other hand, the ranking in the subset for hepatopancreas was RPL7 > RPL3 > RPL8 > ACTB > RPL4 > EF1A > RPL5 > RPL7A > B-TU > UBE2 > PPIB > GAPDH. The pairwise variation assessed by the geNorm program indicates that two reference genes could be sufficient for accurate normalization in both gill and hepatopancreas subsets. Overall, both gill and hepatopancreas subsets recommended ribosomal protein genes (particularly RPL7) as stable references, whereas traditional housekeepers such as ${\beta}-tubulin$ (B-TU) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were ranked as unstable genes. The validation of reference gene selection was confirmed with the quantitative assay of MT transcripts. Conclusions: The present analysis showed the importance of validating reference genes with multiple algorithmic approaches to select genes that are truly stable. Our results indicate that expression stability of a given reference gene could not always have consensus across tissue types. The data from this study could be a good guide for the future design of RT-qPCR studies with respect to metal regulation/detoxification and other related physiologies in this abalone species.

Keywords

References

  1. Amiard JC, Amiard-Triquet C, Barka S, Pellerin J, Rainbow PS. Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. Aquat Toxicol. 2006;76:160-202. https://doi.org/10.1016/j.aquatox.2005.08.015
  2. Andersen CL, Jensen JL, Orntoft TF. 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. 2004;64:5245-50. https://doi.org/10.1158/0008-5472.CAN-04-0496
  3. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611-22. https://doi.org/10.1373/clinchem.2008.112797
  4. Cho YS, Lee SY, Kim KH, Nam YK. Differential modulations of two glyceraldehyde 3-phosphate dehydrogenase mRNAs in response to bacterial and viral challenges in a marine teleost Oplegnathus fasciatus (Perciformes). Fish Shellfish Immunol. 2008;25:472-6. https://doi.org/10.1016/j.fsi.2008.07.007
  5. Cho YS, Kim DS, Nam YK. Isoform-specific response of two GAPDH paralogs during bacterial challenge and metal exposure in mud loach (Misgurnus mizolepis; Cypriniformes) kidney and spleen. J Fish Pathol. 2011;24:269-78. https://doi.org/10.7847/jfp.2011.24.3.269
  6. De Santis C, Smith-Keune C, Jerry DR. Normalizing RT-qPCR data: are we getting the right answers? An appraisal of normalization approaches and internal reference genes from a case study in the finfish Lates calcarifer. Mar Biotechnol. 2011;13:170-80. https://doi.org/10.1007/s10126-010-9277-z
  7. Doak SH, Zair Z. Real-time reverse-transcription polymerase chain reaction: technical considerations for gene expression analysis. In: Parry JM, Parry EM, editors. Genetic toxicology: principles and methods, methods in molecular biology. New York: Springer; 2012. p. 251-70.
  8. Du Y, Zhang L, Xu F, Huang B, Zhang G, Li L. Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR. Fish Shellfish Immunol. 2013;34:939-45. https://doi.org/10.1016/j.fsi.2012.12.007
  9. Fernandes JMO, Mommens M, Hagen O, Babiak I, Solberg C. Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development. Comp Biochem Physiol B. 2008;150:23-32. https://doi.org/10.1016/j.cbpb.2008.01.003
  10. Guenin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L. Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot. 2009;60:487-93. https://doi.org/10.1093/jxb/ern305
  11. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007;8:R19. https://doi.org/10.1186/gb-2007-8-2-r19
  12. Hwang PP, Lee TH, Lin LY. Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms. Am J Physiol Regul Integr Comp Physiol. 2011;301:R28-47. https://doi.org/10.1152/ajpregu.00047.2011
  13. Kim KY, Lee SY, Cho YS, Bang IC, Kim KH, Kim DS, et al. Molecular characterization and mRNA expression during metal exposure and thermal stress of copper/zinc- and manganese-superoxide dismutases in disk abalone, Haliotis discus. Fish Shellfish Immunol. 2007;23:1043-59. https://doi.org/10.1016/j.fsi.2007.04.010
  14. Kim KY, Lee SY, Cho YS, Bang IC, Kim DS, Nam YK. Characterization and phylogeny of two ${\beta}$-cytoskeletal actins from Hemibarbus mylodon (Cyprinidae, Cypriniformes), a threatened fish species in Korea. DNA Seq. 2008;19:87-97. https://doi.org/10.1080/10425170701445691
  15. Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonak J, Lind K, et al. The real-time polymerase chain reaction. Mol Asp Med. 2006;27:95-125. https://doi.org/10.1016/j.mam.2005.12.007
  16. Le DT, Aldrich DL, Valliyodan B, Watanabe Y, Ha CV. Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions. PLoS One. 2012;7:e46487. https://doi.org/10.1371/journal.pone.0046487
  17. Lee SY, Kim DS, Nam YK. Genomic organization, tissue distribution and developmental expression of glyceraldehyde 3-phosphate dehydrogenase isoforms in mud loach Misgurnus mizolepis. Fish Aquat Sci. 2013;16:291-301.
  18. Li R, Shen Y. An old method facing a new challenge: re-visiting housekeeping proteins as internal reference control for neuroscience research. Life Sci. 2013;92:747-51. https://doi.org/10.1016/j.lfs.2013.02.014
  19. Lopez-Landavery EA, Portillo-Lopez A, Gallardo-Escarate C, Rio-Portilla MAD. Selection of reference genes as internal controls for gene expression in tissues of red abalone Haliotis rufescens (Mollusca, Vetigastropoda; Swainson, 1822). Gene. 2014;549:258-65. https://doi.org/10.1016/j.gene.2014.08.002
  20. Mao H, Wang DH, Yang WX. Involvement of metallothionein in the development of aquatic invertebrate. Aquatic Toxicol. 2012;110-111:208-13. https://doi.org/10.1016/j.aquatox.2012.01.018
  21. Overgard AC, Nerland AH, Patel S. Evaluation of potential reference genes for real time RT-PCR studies in Atlantic halibut (Hippoglossus hippoglossus L.); during development, in tissues of healthy and NNV-injected fish, and in anterior kidney leucocytes. BMC Mol Biol. 2010;11:36. https://doi.org/10.1186/1471-2199-11-36
  22. Park CJ, Kim SY. Abalone aquaculture in Korea. J Shellfish Res. 2013;32:17-9. https://doi.org/10.2983/035.032.0104
  23. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004;26:509-15. https://doi.org/10.1023/B:BILE.0000019559.84305.47
  24. Qiu R, Sun B, Fang S, Sun L, Liu X. Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai. Chin J Oceanol Limnol. 2013;31:421-30. https://doi.org/10.1007/s00343-013-2221-0
  25. Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 2004;313:856-62. https://doi.org/10.1016/j.bbrc.2003.11.177
  26. Rainbow PS. Trace metal bioaccumulation: models, metabolic availability and toxicity. Environ Intl. 2007;33:576-82. https://doi.org/10.1016/j.envint.2006.05.007
  27. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008;3:1101-08. https://doi.org/10.1038/nprot.2008.73
  28. Shen GM, Jiang HB, Wang XN, Wang JJ. Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae). BMC Mol Biol. 2010;11:76. https://doi.org/10.1186/1471-2199-11-76
  29. Silva-Aciares F, Zapata M, Tournois J, Moraga D, Riquelme C. Identification of genes expressed in juvenile Haliotis rufescens in response to different copper concentrations in the north of Chile under controlled conditions. Mar Pollut Bull. 2011;62:2671-80. https://doi.org/10.1016/j.marpolbul.2011.09.023
  30. Silver N, Best S, Jiang J, Thein SL. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol. 2006;7:33. https://doi.org/10.1186/1471-2199-7-33
  31. Song Y, Choi MS, Lee JY, Jang DJ. Regional background concentrations of heavy metals (Cr, Co, Ni, Cu, Zn, Pb) in coastal sediments of the South Sea of Korea. Sci Total Environ. 2014;482-483:80-91. https://doi.org/10.1016/j.scitotenv.2014.02.068
  32. Taylor DA, Thompson EL, Nair SV, Raftos DA. Differential effects of metal contamination on the transcript expression of immune- and stress-response genes in the Sydney rock oyster, Saccostrea glomerata. Environ Pollt. 2013;178:65-71. https://doi.org/10.1016/j.envpol.2013.02.027
  33. Turkmen M, Turkmen A, Tepe Y, Ates A, Gokkus K. Determination of metal contaminations in sea foods from Marmara, Aegean and Mediterranean seas: twelve fish species. Food Chem. 2008;108:794-800. https://doi.org/10.1016/j.foodchem.2007.11.025
  34. Udvardi MK, Czechowski T, Scheible WR. Eleven golden rules of quantitative RT-PCR. Plant Cell. 2008;20:1736-7. https://doi.org/10.1105/tpc.108.061143
  35. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:1-11. research0034.
  36. Wan Q, Whang I, Choi CY, Lee JS, Lee J. Validation of housekeeping genes as internal controls for studying biomarkers of endocrine-disrupting chemicals in disk abalone by real-time PCR. Comp Biochem Physiol C. 2011;153:259-68.
  37. Wang WX, Rainbow PS. Influence of metal exposure history on trace metal uptake and accumulation by marine invertebrates. Ecotoxicol Environ Saf. 2005;61:145-59. https://doi.org/10.1016/j.ecoenv.2005.01.008
  38. Yuan M, Lu Y, Zhu X, Wan H, Shakeel M, Zhan S, et al. Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR. PLoS One. 2014;9:e86503. https://doi.org/10.1371/journal.pone.0086503
  39. Zhang Z, Hu J. Development and validation of endogenous reference genes for expression profiling of medaka (Oryzias latipes) exposed to endocrine disrupting chemicals by quantitative real-time RT-PCR. Toxicol Sci. 2007;95:356-68. https://doi.org/10.1093/toxsci/kfl161
  40. Zheng WJ, Sun L. Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder (Paralichthys olivaceus). Fish Shellfish Immunol. 2011;30:638-45. https://doi.org/10.1016/j.fsi.2010.12.014
  41. Zhu L, Altmann SW. mRNA and 18S-RNA coapplication-reverse transcription for quantitative gene expression analysis. Anal Biochem. 2005;345:102-9. https://doi.org/10.1016/j.ab.2005.07.028

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