Validation of Reference Genes for Quantitative RT-qPCR Studies of Gene Expression in Atlantic Cod (Gadus Morhua L.) During Temperature Stress

Overview

Journal BMC Res Notes

Publisher Biomed Central

Specialties Biology
General Medicine

Date 2011 Apr 7

PMID 21466674

Citations 15

Authors

Ingrid A Aursnes

Anne L Rishovd

Hans E Karlsen

Tor Gjoen

Affiliations

Soon will be listed here.

Abstract

Background: One important physiological response to environmental stress in animals is change in gene expression. To obtain reliable data from gene expression studies using RT-qPCR it is important to evaluate a set of possible reference genes as normalizers for expression. The expression of these candidate genes should be analyzed in the relevant tissues during normal and stressed situations. To find suitable reference genes it was crucial that the genes were stably expressed also during a situation of physiological stress. For poikilotermic animals like cod, changes in temperature are normal, but if the changes are faster than physiological compensation, the animals respond with typical stress responses. It has previously been shown that Atlantic cod show stress responses when elevation of water temperature is faster than 1 degree/day, for this reason we chose hyperthermia as stress agent for this experiment.

Findings: We here describe the expression of eight candidate reference genes from Atlantic cod (Gadus morhua l.) and their stability during thermal stress (temperature elevation of one degree C/day for 5 days). The genes investigated were: Eukaryotic elongation factor 1 alpha, ef1a; 18s ribosomal RNA; 18s, Ubiquitin conjugate protein; ubiq, cytoskeletal beta-actin; actb, major histcompatibility complex I; MHC-I light chain, beta-2 -microglobulin; b2m, cytoskeletal alpha-tubulin; tba1c, acidic ribosomal phosphoprotein; rplp1, glucose-6-phosphate dehydrogenase; g6pd. Their expression were analyzed in 6 tissues (liver, head kidney, intestine, spleen, heart and gills) from cods exposed to elevated temperature and compared to a control group. Although there were variations between tissues with respect to reference gene stability, four transcripts were more consistent than the others: ubiq, ef1a, 18s and rplp1. We therefore used these to analyze the expression of stress related genes (heat shock proteins) induced during hyperthermia. We found that both transcripts were significantly upregulated in several tissues in fish exposed to increased temperature.

Conclusion: This is the first study comparing reference genes for RT-qPCR analyses of expression during hyperthermia in Atlantic cod. ef1a, 18s, rplp1 and ubiq transcripts were found to be well suited as reference genes during these experimental conditions.

Citing Articles

Gene Expression and Phenotypic Assessment of Egg Quality across Developmental Stages of Atlantic Cod throughout the Spawning Season.

Fernandez Miguez M, Presa P, Puvanendran V, Tveiten H, Hansen O, Perez M Int J Mol Sci. 2024; 25(13).

PMID: 39000593 PMC: 11242223. DOI: 10.3390/ijms25137488.

Effects of Stocking Density on the Growth Performance, Physiological Parameters, Antioxidant Status and Lipid Metabolism of in the Integrated Rice-Fish Farming System.

Diao W, Jia R, Hou Y, Dong Y, Li B, Zhu J Animals (Basel). 2023; 13(11).

PMID: 37889625 PMC: 10251942. DOI: 10.3390/ani13111721.

RPLP1 Is Up-Regulated in Human Adenomyosis and Endometrial Adenocarcinoma Epithelial Cells and Is Essential for Cell Survival and Migration In Vitro.

Peterson R, Minchella P, Cui W, Graham A, Nothnick W Int J Mol Sci. 2023; 24(3).

PMID: 36769010 PMC: 9917350. DOI: 10.3390/ijms24032690.

High-Temperature Stress Effect on the Red Cusk-Eel () Liver: Transcriptional Modulation and Oxidative Stress Damage.

Dettleff P, Zuloaga R, Fuentes M, Gonzalez P, Aedo J, Estrada J Biology (Basel). 2022; 11(7).

PMID: 36101373 PMC: 9312335. DOI: 10.3390/biology11070990.

60S acidic ribosomal protein P1 (RPLP1) is elevated in human endometriotic tissue and in a murine model of endometriosis and is essential for endometriotic epithelial cell survival in vitro.

Alali Z, Graham A, Swan K, Flyckt R, Falcone T, Cui W Mol Hum Reprod. 2020; 26(1):53-64.

PMID: 31899515 PMC: 8204708. DOI: 10.1093/molehr/gaz065.

References

Jorgensen S, Kleveland E, Grimholt U, Gjoen T . Validation of reference genes for real-time polymerase chain reaction studies in Atlantic salmon. Mar Biotechnol (NY). 2006; 8(4):398-408. DOI: 10.1007/s10126-005-5164-4. View

Olsvik P, Softeland L, Lie K . Selection of reference genes for qRT-PCR examination of wild populations of Atlantic cod Gadus morhua. BMC Res Notes. 2008; 1:47. PMC: 2527504. DOI: 10.1186/1756-0500-1-47. View

Lindquist S, Craig E . The heat-shock proteins. Annu Rev Genet. 1988; 22:631-77. DOI: 10.1146/annurev.ge.22.120188.003215. View

Pfaffl M, Tichopad A, Prgomet C, Neuvians T . Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004; 26(6):509-15. DOI: 10.1023/b:bile.0000019559.84305.47. View

Saele O, Nordgreen A, Hamre K, Olsvik P . Evaluation of candidate reference genes in Q-PCR studies of Atlantic cod (Gadus morhua) ontogeny, with emphasis on the gastrointestinal tract. Comp Biochem Physiol B Biochem Mol Biol. 2008; 152(1):94-101. DOI: 10.1016/j.cbpb.2008.10.002. View

Klinkert B, Narberhaus F . Microbial thermosensors. Cell Mol Life Sci. 2009; 66(16):2661-76. PMC: 11115684. DOI: 10.1007/s00018-009-0041-3. View

Manchado M, Salas-Leiton E, Infante C, Ponce M, Asensio E, Crespo A . Molecular characterization, gene expression and transcriptional regulation of cytosolic HSP90 genes in the flatfish Senegalese sole (Solea senegalensis Kaup). Gene. 2008; 416(1-2):77-84. DOI: 10.1016/j.gene.2008.03.007. View

Diller K . Stress protein expression kinetics. Annu Rev Biomed Eng. 2006; 8:403-24. DOI: 10.1146/annurev.bioeng.7.060804.100449. View

Andersen C, Jensen J, Orntoft T . 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(15):5245-50. DOI: 10.1158/0008-5472.CAN-04-0496. View

10.

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

11.

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

12.

Pfaffl M, Horgan G, Dempfle L . Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002; 30(9):e36. PMC: 113859. DOI: 10.1093/nar/30.9.e36. View

13.

Ingerslev H, Pettersen E, Jakobsen R, Petersen C, Wergeland H . Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.). Mol Immunol. 2005; 43(8):1194-201. DOI: 10.1016/j.molimm.2005.07.009. View

14.

McCurley A, Callard G . Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment. BMC Mol Biol. 2008; 9:102. PMC: 2588455. DOI: 10.1186/1471-2199-9-102. View

15.

Dheda K, Huggett J, Bustin S, Johnson M, Rook G, Zumla A . Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques. 2004; 37(1):112-4, 116, 118-9. DOI: 10.2144/04371RR03. View

16.

Yamashita M, Hirayoshi K, Nagata K . Characterization of multiple members of the HSP70 family in platyfish culture cells: molecular evolution of stress protein HSP70 in vertebrates. Gene. 2004; 336(2):207-18. DOI: 10.1016/j.gene.2004.04.023. View

17.

Kampinga H, Hageman J, Vos M, Kubota H, Tanguay R, Bruford E . Guidelines for the nomenclature of the human heat shock proteins. Cell Stress Chaperones. 2008; 14(1):105-11. PMC: 2673902. DOI: 10.1007/s12192-008-0068-7. View

18.

Fernandes J, 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 Biochem Mol Biol. 2008; 150(1):23-32. DOI: 10.1016/j.cbpb.2008.01.003. View

19.

Perez-Casanova J, Rise M, Dixon B, Afonso L, Hall J, Johnson S . The immune and stress responses of Atlantic cod to long-term increases in water temperature. Fish Shellfish Immunol. 2008; 24(5):600-9. DOI: 10.1016/j.fsi.2008.01.012. View

20.

Kapp L, Lorsch J . The molecular mechanics of eukaryotic translation. Annu Rev Biochem. 2004; 73:657-704. DOI: 10.1146/annurev.biochem.73.030403.080419. View