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Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies

Suzanne Vanhauwaert (UGent) , Gert Van Peer (UGent) , Ali Rihani (UGent) , Els Janssens (UGent) , Pieter Rondou (UGent) , Steve Lefever (UGent) , Anne De Paepe (UGent) , Paul Coucke (UGent) , Franki Speleman (UGent) , Jo Vandesompele (UGent) , et al.
(2014) PLOS ONE. 9(10).
Author
Organization
Project
Bioinformatics: from nucleotids to networks (N2N)
Abstract
The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions. In this study, we evaluated a novel normalization method in zebrafish using expressed repetitive elements (ERE) as reference targets, instead of specific protein coding mRNA targets. We assessed and compared the expression stability of a number of EREs to that of commonly used zebrafish reference genes in a diverse set of experimental conditions including a developmental time series, a set of different organs from adult fish and different treatments of zebrafish embryos including morpholino injections and administration of chemicals. Using geNorm and rank aggregation analysis we demonstrated that EREs have a higher overall expression stability compared to the commonly used reference genes. Moreover, we propose a limited set of ERE reference targets (hatn10, dna15ta1 and loopern4), that show stable expression throughout the wide range of experiments in this study, as strong candidates for inclusion as reference targets for qPCR normalization in future zebrafish expression studies. Our applied strategy to find and evaluate candidate expressed repeat elements for RT-qPCR data normalization has high potential to be used also for other species.
Keywords
POLYMERASE-CHAIN-REACTION, HOUSEKEEPING GENES, REAL-TIME PCR, EMBRYOS, GLUT10, RIBOSOMAL-RNA, MESSENGER, QUANTIFICATION, VALIDATION, INAPPROPRIATE

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Citation

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Chicago
Vanhauwaert, Suzanne, Gert Van Peer, Ali Rihani, Els Janssens, Pieter Rondou, Steve Lefever, Anne De Paepe, et al. 2014. “Expressed Repeat Elements Improve RT-qPCR Normalization Across a Wide Range of Zebrafish Gene Expression Studies.” Plos One 9 (10).
APA
Vanhauwaert, Suzanne, Van Peer, G., Rihani, A., Janssens, E., Rondou, P., Lefever, S., De Paepe, A., et al. (2014). Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies. PLOS ONE, 9(10).
Vancouver
1.
Vanhauwaert S, Van Peer G, Rihani A, Janssens E, Rondou P, Lefever S, et al. Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies. PLOS ONE. 2014;9(10).
MLA
Vanhauwaert, Suzanne, Gert Van Peer, Ali Rihani, et al. “Expressed Repeat Elements Improve RT-qPCR Normalization Across a Wide Range of Zebrafish Gene Expression Studies.” PLOS ONE 9.10 (2014): n. pag. Print.
@article{5822567,
  abstract     = {The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions. In this study, we evaluated a novel normalization method in zebrafish using expressed repetitive elements (ERE) as reference targets, instead of specific protein coding mRNA targets. We assessed and compared the expression stability of a number of EREs to that of commonly used zebrafish reference genes in a diverse set of experimental conditions including a developmental time series, a set of different organs from adult fish and different treatments of zebrafish embryos including morpholino injections and administration of chemicals. Using geNorm and rank aggregation analysis we demonstrated that EREs have a higher overall expression stability compared to the commonly used reference genes. Moreover, we propose a limited set of ERE reference targets (hatn10, dna15ta1 and loopern4), that show stable expression throughout the wide range of experiments in this study, as strong candidates for inclusion as reference targets for qPCR normalization in future zebrafish expression studies. Our applied strategy to find and evaluate candidate expressed repeat elements for RT-qPCR data normalization has high potential to be used also for other species.},
  articleno    = {e109091},
  author       = {Vanhauwaert, Suzanne and Van Peer, Gert and Rihani, Ali and Janssens, Els and Rondou, Pieter and Lefever, Steve and De Paepe, Anne and Coucke, Paul and Speleman, Franki and Vandesompele, Jo and Willaert, Andy},
  issn         = {1932-6203},
  journal      = {PLOS ONE},
  keyword      = {POLYMERASE-CHAIN-REACTION,HOUSEKEEPING GENES,REAL-TIME PCR,EMBRYOS,GLUT10,RIBOSOMAL-RNA,MESSENGER,QUANTIFICATION,VALIDATION,INAPPROPRIATE},
  language     = {eng},
  number       = {10},
  pages        = {10},
  title        = {Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies},
  url          = {http://dx.doi.org/10.1371/journal.pone.0109091},
  volume       = {9},
  year         = {2014},
}

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