Fish Physiology and Biochemistry

, Volume 42, Issue 1, pp 125–135 | Cite as

Evaluation of housekeeping genes as references for quantitative real-time PCR analysis of gene expression in the murrel Channa striatus under high-temperature stress

Article

Abstract

Quantitative real-time polymerase chain reaction is the most advanced method of quantifying gene expression studies; however, the significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. In the present study, expression analysis of six different constitutively expressed genes viz. 18S ribosomal RNA, glyceraldehyde-3-phosphate dehydrogenase (gapdh), beta actin (βactin), ribosomal binding protein L13, tubulin and TATA-box-binding protein (tbp) were carried out to test their efficacy as reference genes in three different tissues, namely liver, gill and muscle of murrel Channa striatus exposed to high temperature for variable time periods. The stability and suitability of the genes were determined by using bioinformatic tools: GeNorm, NormFinder and BestKeeper. Based on the results, tub/βactin could be used as the reference genes for liver and gill tissues and βactin/gapdh could be the reference genes for muscle tissues in Channa striatus under both short- and long-term thermal stress.

Keywords

Reference genes Quantitative real-time PCR Normalization Channa striatus Thermal stress 

Supplementary material

10695_2015_123_MOESM1_ESM.pdf (25 kb)
Supplementary data 1 NCBI sequence information used for designing primers for amplification of different genes. Supplementary material 1 (PDF 24 kb)
10695_2015_123_MOESM2_ESM.pdf (294 kb)
Supplementary data 2 Standard curves showing (A) tbp, (B) βactin, (C) gapdh, (D) rbpl13, (E) tub, (F) 18SrRNA. Supplementary material 2 (PDF 293 kb)
10695_2015_123_MOESM3_ESM.pdf (35 kb)
Supplementary data 3 Descriptive statistics of the six candidate reference genes based on their cycle threshold (Ct) values as calculated by BestKeeper. Supplementary material 3 (PDF 35 kb)

References

  1. Andersen CL, Jensen JL, Orntoft 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 64:5245–5250CrossRefPubMedGoogle Scholar
  2. Arukwe A (2006) Toxicological housekeeping genes: do they really keep the house? Environ Sci Technol 40:7944–7949CrossRefPubMedGoogle Scholar
  3. Aursnes IA, Rishovd AL, Karlsen HE, Gjoen T (2011) Validation of reference genes for quantitative RT-qPCR studies of gene expression in Atlantic cod (Gadus morhua l.) during temperature stress. BMC Res Notes 4:104PubMedCentralCrossRefPubMedGoogle Scholar
  4. Barber RD, Harmer DW, Coleman RA, Clark BJ (2005) GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiol Genom 21:389–395CrossRefGoogle Scholar
  5. Bolton TF, Havenhand JN (2005) Physiological acclimation to decreased water temperature and the relative importance of water viscosity in determining the feeding performance of larvae of a serpulid polychaete. J Plankton Res 27:875–879CrossRefGoogle Scholar
  6. Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193CrossRefPubMedGoogle Scholar
  7. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622CrossRefPubMedGoogle Scholar
  8. Deloffre LA, Andrade A, Filipe AI, Canario AV (2012) Reference genes to quantify gene expression during oogenesis in a teleost fish. Gene 506:69–75CrossRefPubMedGoogle Scholar
  9. Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GAW, Zumla A (2005) The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 344:141–143CrossRefPubMedGoogle Scholar
  10. Feng L, Yu Q, Li X, Ning X, Wang J, Zou J, Zhang L, Wang S, Hu J, Hu X, Bao Z (2013) Identification of reference genes for qRT-PCR analysis in Yesso scallop Patinopecten yessoensis. PLoS ONE 8:e75609PubMedCentralCrossRefPubMedGoogle Scholar
  11. Filby AL, Tyler CR (2007) Appropriate ‘housekeeping’ genes for use in expression profiling the effects of environmental estrogens in fish. BMC Mol Biol 8:10PubMedCentralCrossRefPubMedGoogle Scholar
  12. Hibbeler S, Scharsack JP, Becker S (2008) Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus. BMC Mol Biol 9:8CrossRefGoogle Scholar
  13. Infante C, Matsuoka MP, Asensio E, Canavate JP, Reith M, Manchado M (2008) Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR. BMC Mol Biol 9:28PubMedCentralCrossRefPubMedGoogle Scholar
  14. Ingerslev HC, Pettersen EF, Jakobsen RA, Petersen CB, Wergeland HI (2006) Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.). Mol Immunol 43:1194–1201CrossRefPubMedGoogle Scholar
  15. IPCC (2007) Fourth Assessment Report—Climate Change, Synthesis Report. Geneva, SwitzerlandGoogle Scholar
  16. Jee BY, Kim KH, Park SI, Kim YC (2000) A new strain of Cryptocaryon irritans from the cultured olive flounder Paralichthys olivaceus. Dis Aquat Organ 43:211–215CrossRefPubMedGoogle Scholar
  17. Kapila N, Kishore A, Sodhi M, Sharma A, Kumar P, Mohanty AK, Jerath T, Mukesh M (2013) Identification of appropriate reference genes for qRT-PCR analysis of heat-stressed mammary epithelial cells in riverine buffaloes (Bubalus bubalis). ISRN Biotechnol 2013:735053PubMedCentralCrossRefPubMedGoogle Scholar
  18. Kreuzer KA, Lass U, Landt O, Nitsche A, Laser J, Ellerbrok H, Pauli G, Huhn D, Schmidt CA (1999) Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of ß-actin transcripts as quantitative reference. Clin Chem 45:297–300PubMedGoogle Scholar
  19. Kubista M, Andrade JM, Bengtsson M, Forootan A, Jonák J, Lind K et al (2006) The real-time polymerase chain reaction. Mol Aspects Med 27:95e125CrossRefGoogle Scholar
  20. Mane VP, Heuer MA, Hillyer P, Navarro MB, Rabin RL (2008) Systematic method for determining an ideal housekeeping gene for real-time PCR analysis. J Biomol Tech 19:342–347PubMedCentralPubMedGoogle Scholar
  21. Maroufi A, Bockstaele EV, De Loose M (2010) Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol 11:15PubMedCentralCrossRefPubMedGoogle Scholar
  22. McCurley AT, Callard GV (2008) Characterization of housekeeping genes in zebrafish: male–female differences and effects of tissue type, developmental stage and chemical treatment. BMC Mol Biol 9:102PubMedCentralCrossRefPubMedGoogle Scholar
  23. Mohanty S, Mahanty A, Yadav RP, Purohit GK, Mohanty BN, Mohanty BP (2014) Atri hot spring—a natural ecosystem for global warming research. Int J Geol Earth Environ Sci 4:85–90Google Scholar
  24. Mohindra V, Tripathi RK, Singh A, Singh RK, Lal KK (2014) Identification of candidate reference genes for quantitative expression analysis by real-time PCR for hypoxic stress in Indian catfish, Clarias batrachus (Linnaeus, 1758). Int Aquat Res 6:1–12CrossRefGoogle Scholar
  25. Mubiana VK, Blust R (2007) Effects of temperature on scope for growth and accumulation of Cd Co, Cu and Pb by the marine bivalve Mytilus edulis. Mar Environ Res 63:219–235CrossRefPubMedGoogle Scholar
  26. Olsvik PA, Lie KK, Jordal AEO, Nilsen TO, Hordvik I (2005) Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon. BMC Mol Biol 6:21PubMedCentralCrossRefPubMedGoogle Scholar
  27. Ozturk ZN, Talame V, Deyholos M, Michalowski CB, Galbraith DW, Gozukirmizi N, Tuberosa R, Bohnert HJ (2002) Monitoring large-scale changes in transcript abundance in drought- and salt-stressed barley. Plant Mol Biol 48:551–573CrossRefGoogle Scholar
  28. 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. Biotechnol Lett 26:509–515CrossRefPubMedGoogle Scholar
  29. Purohit GK, Mahanty A, Suar M, Sharma AP, Mohanty BP, Mohanty S (2014) Investigating HSP gene expression in liver of Channa striatus under heat stress for understanding the upper thermal acclimation. Biomed Res Int 381719:1–10CrossRefGoogle Scholar
  30. Setiawan AN, Lokman PM (2010) The use of reference gene selection programs to study the silvering transformation in a freshwater eel Anguilla australis: a cautionary tale. BMC Mol Biol 11:75PubMedCentralCrossRefPubMedGoogle Scholar
  31. Silva RLO, Silva MD, Neto JRCF, de Nardi CH, Moutinho S (2014) Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane. Sci World J 2014:357052Google Scholar
  32. Small BC, Murdock CA, Bilodeau-Bourgeois AL, Peterson BC, Waldbieser GC (2008) Stability of reference genes for real-time PCR analyses in channel catfish (Ictalurus punctatus) tissues under varying physiological conditions. Comp Biochem Physiol B: Biochem Mol Biol 151:296–304CrossRefGoogle Scholar
  33. Tang R, Dodd A, Lai D, McNabb WC, Love DR (2007) Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization. Acta Biochim Biophys Sin 39:384–390CrossRefPubMedGoogle Scholar
  34. Tarze A, Deniaud A, LeBras M, Maillier E, Molle D, Larochette N, Zamzami N, Jan G, Kroemer G, Brenner C (2007) GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization. Oncogene 26:2606–2620CrossRefPubMedGoogle Scholar
  35. Urbatzka R, Galante Oliveira S, Rocha E, Castro LF, Cunha I (2013) Normalization strategies for gene expression studies by real-time PCR in a marine fish species, Scophthalmus maximus. Mar Genom 10:17–25CrossRefGoogle Scholar
  36. 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 3:0034CrossRefGoogle Scholar
  37. Vandesompele J, Kubista M, Pfaffl MW (2009) Reference gene validation software for improved normalization. In: Logan J, Edwards K, Saunders N, Norfolk UK (eds) Real-time PCR: current technology and applications. Caister Academic Press, England, pp 47–64Google Scholar
  38. Williams TD, Gensberg K, Minchin SD, Chipman JK (2003) A DNA expression array to detect toxic stress response in European flounder (Platichthys flesus). Aquat Toxicol 65:141–157CrossRefPubMedGoogle Scholar
  39. Ye X, Zhang L, Dong H, Tian Y, Lao H, Bai J, Yu L (2010) Validation of reference genes of grass carp Ctenopharyngodon idellus for the normalization of quantitative real-time PCR. Biotechnol Lett 32:1031–1038CrossRefPubMedGoogle Scholar
  40. Zala D, Hinckelmann MV, Yu H, da Cunha MML, Liot G, Cordelieres FP, Marco S, Saudou F (2013) Vesicular glycolysis provides on-board energy for fast axonal transport. Cell 152:479–491CrossRefPubMedGoogle Scholar
  41. Zhang BC, Sun L, Xiao ZZ, Hu YH (2014) Quantitative real time RT-PCR study of pathogen induced gene expression in rock bream (Oplegnathus fasciatus): internal control for data normalization. Mar Genom 15:75–84CrossRefGoogle Scholar
  42. Zheng WJ, Sun L (2011) Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder (Paralichthys olivaceus). Fish Shellfish Immunol 30:638–645CrossRefPubMedGoogle Scholar
  43. Zhou RX, Meng T, Meng HB, Cheng DX, Bin SY, Cheng J, Fu GH, Chu WY, Zhang JS (2010) Selection of reference genes in transcription analysis of gene expression of the mandarin fish, Siniperca chuasti. Dongwuxue Yanjiu 31:141–146PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  1. 1.KIIT School of BiotechnologyKIIT UniversityBhubaneswarIndia
  2. 2.Fishery Resource and Environmental Management DivisionICAR- Central Inland Fisheries Research InstituteBarrackpore, KolkataIndia

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