Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions
Some mammalian reference genes, which are widely used to normalize the qRT-PCR, could not be used for this purpose due to its high expression variation. The normalization with false reference genes leads to misinterpretation of results. The silversides (Odontesthes spp.) has been used as models for...
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2018-03-01
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doaj-964d7e5f12684664be3da058fb810d892020-11-24T22:11:47ZengFrontiers Media S.A.Frontiers in Genetics1664-80212018-03-01910.3389/fgene.2018.00075322967Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental ConditionsTony L. R. Silveira0William B. Domingues1Mariana H. Remião2Lucas Santos3Bruna Barreto4Ingrid M. Lessa5Antonio Sergio Varela Junior6Diego Martins Pires7Carine Corcini8Tiago Collares9Fabiana K. Seixas10Ricardo B. Robaldo11Vinicius F. Campos12Laboratory of Structural Genomics, Biotechnology Graduate Program, Federal University of Pelotas, Pelotas, BrazilLaboratory of Structural Genomics, Biotechnology Graduate Program, Federal University of Pelotas, Pelotas, BrazilLaboratory of Structural Genomics, Biotechnology Graduate Program, Federal University of Pelotas, Pelotas, BrazilLaboratory of Structural Genomics, Biotechnology Graduate Program, Federal University of Pelotas, Pelotas, BrazilLaboratory of Structural Genomics, Biotechnology Graduate Program, Federal University of Pelotas, Pelotas, BrazilLaboratory of Structural Genomics, Biotechnology Graduate Program, Federal University of Pelotas, Pelotas, BrazilInstitute of Biological Sciences, Federal University of Rio Grande, Rio Grande, BrazilVeterinary Faculty, Federal University of Pelotas, Pelotas, BrazilVeterinary Faculty, Federal University of Pelotas, Pelotas, BrazilLaboratory of Cancer Biotechnology, Biotechnology Graduate Program, Federal University of Pelotas, Pelotas, BrazilLaboratory of Cancer Biotechnology, Biotechnology Graduate Program, Federal University of Pelotas, Pelotas, BrazilLaboratory of Physiology, Institute of Biology, Federal University of Pelotas, Pelotas, BrazilLaboratory of Structural Genomics, Biotechnology Graduate Program, Federal University of Pelotas, Pelotas, BrazilSome mammalian reference genes, which are widely used to normalize the qRT-PCR, could not be used for this purpose due to its high expression variation. The normalization with false reference genes leads to misinterpretation of results. The silversides (Odontesthes spp.) has been used as models for evolutionary, osmoregulatory and environmental pollution studies but, up to now, there are no studies about reference genes in any Odontesthes species. Furthermore, many studies on silversides have used reference genes without previous validations. Thus, present study aimed to was to clone and sequence potential reference genes, thereby identifying the best ones in Odontesthes humensis considering different tissues, ages and conditions. For this purpose, animals belonging to three ages (adults, juveniles, and immature) were exposed to control, Roundup®, and seawater treatments for 24 h. Blood samples were subjected to flow-cytometry and other collected tissues to RNA extraction; cDNA synthesis; molecular cloning; DNA sequencing; and qRT-PCR. The candidate genes tested included 18s, actb, ef1a, eif3g, gapdh, h3a, atp1a, and tuba. Gene expression results were analyzed using five algorithms that ranked the candidate genes. The flow-cytometry data showed that the environmental challenges could trigger a systemic response in the treated fish. Even during this systemic physiological disorder, the consensus analysis of gene expression revealed h3a to be the most stable gene expression when only the treatments were considered. On the other hand, tuba was the least stable gene in the control and gapdh was the least stable in both Roundup® and seawater groups. In conclusion, the consensus analyses of different tissues, ages, and treatments groups revealed that h3a is the most stable gene whereas gapdh and tuba are the least stable genes, even being considered two constitutive genes.http://journal.frontiersin.org/article/10.3389/fgene.2018.00075/fullalgorithmexpressionfishgenenormalizationreal time PCR |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Tony L. R. Silveira William B. Domingues Mariana H. Remião Lucas Santos Bruna Barreto Ingrid M. Lessa Antonio Sergio Varela Junior Diego Martins Pires Carine Corcini Tiago Collares Fabiana K. Seixas Ricardo B. Robaldo Vinicius F. Campos |
spellingShingle |
Tony L. R. Silveira William B. Domingues Mariana H. Remião Lucas Santos Bruna Barreto Ingrid M. Lessa Antonio Sergio Varela Junior Diego Martins Pires Carine Corcini Tiago Collares Fabiana K. Seixas Ricardo B. Robaldo Vinicius F. Campos Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions Frontiers in Genetics algorithm expression fish gene normalization real time PCR |
author_facet |
Tony L. R. Silveira William B. Domingues Mariana H. Remião Lucas Santos Bruna Barreto Ingrid M. Lessa Antonio Sergio Varela Junior Diego Martins Pires Carine Corcini Tiago Collares Fabiana K. Seixas Ricardo B. Robaldo Vinicius F. Campos |
author_sort |
Tony L. R. Silveira |
title |
Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions |
title_short |
Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions |
title_full |
Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions |
title_fullStr |
Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions |
title_full_unstemmed |
Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions |
title_sort |
evaluation of reference genes to analyze gene expression in silverside odontesthes humensis under different environmental conditions |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Genetics |
issn |
1664-8021 |
publishDate |
2018-03-01 |
description |
Some mammalian reference genes, which are widely used to normalize the qRT-PCR, could not be used for this purpose due to its high expression variation. The normalization with false reference genes leads to misinterpretation of results. The silversides (Odontesthes spp.) has been used as models for evolutionary, osmoregulatory and environmental pollution studies but, up to now, there are no studies about reference genes in any Odontesthes species. Furthermore, many studies on silversides have used reference genes without previous validations. Thus, present study aimed to was to clone and sequence potential reference genes, thereby identifying the best ones in Odontesthes humensis considering different tissues, ages and conditions. For this purpose, animals belonging to three ages (adults, juveniles, and immature) were exposed to control, Roundup®, and seawater treatments for 24 h. Blood samples were subjected to flow-cytometry and other collected tissues to RNA extraction; cDNA synthesis; molecular cloning; DNA sequencing; and qRT-PCR. The candidate genes tested included 18s, actb, ef1a, eif3g, gapdh, h3a, atp1a, and tuba. Gene expression results were analyzed using five algorithms that ranked the candidate genes. The flow-cytometry data showed that the environmental challenges could trigger a systemic response in the treated fish. Even during this systemic physiological disorder, the consensus analysis of gene expression revealed h3a to be the most stable gene expression when only the treatments were considered. On the other hand, tuba was the least stable gene in the control and gapdh was the least stable in both Roundup® and seawater groups. In conclusion, the consensus analyses of different tissues, ages, and treatments groups revealed that h3a is the most stable gene whereas gapdh and tuba are the least stable genes, even being considered two constitutive genes. |
topic |
algorithm expression fish gene normalization real time PCR |
url |
http://journal.frontiersin.org/article/10.3389/fgene.2018.00075/full |
work_keys_str_mv |
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