Validation of reference genes for expression studies during craniofacial development in arctic charr.

Arctic charr (Salvelinus alpinus) is a highly polymorphic species and in Lake Thingvallavatn, Iceland, four phenotypic morphs have evolved. These differences in morphology, especially in craniofacial structures are already apparent during embryonic development, indicating that genes important in the...

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Autores principales:	Ehsan Pashay Ahi, Jóhannes Guðbrandsson, Kalina H Kapralova, Sigríður R Franzdóttir, Sigurður S Snorrason, Valerie H Maier, Zophonías O Jónsson
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/3016dd142811451294a002ebd90fcf9a
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spelling	oai:doaj.org-article:3016dd142811451294a002ebd90fcf9a2021-11-18T07:41:50ZValidation of reference genes for expression studies during craniofacial development in arctic charr.1932-620310.1371/journal.pone.0066389https://doaj.org/article/3016dd142811451294a002ebd90fcf9a2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23785496/?tool=EBIhttps://doaj.org/toc/1932-6203Arctic charr (Salvelinus alpinus) is a highly polymorphic species and in Lake Thingvallavatn, Iceland, four phenotypic morphs have evolved. These differences in morphology, especially in craniofacial structures are already apparent during embryonic development, indicating that genes important in the formation of the craniofacial features are expressed differentially between the morphs. In order to generate tools to examine these expression differences in Arctic charr, the aim of the present study was to identify reference genes for quantitative real-time PCR (qPCR). The specific aim was to select reference genes which are able to detect very small expression differences among different morphs. We selected twelve candidate reference genes from the literature, identified corresponding charr sequences using data derived from transcriptome sequencing (RNA-seq) and examined their expression using qPCR. Many of the candidate reference genes were found to be stably expressed, yet their quality-rank as reference genes varied considerably depending on the type of analysis used. In addition to commonly used software for reference gene validation, we used classical statistics to evaluate expression profiles avoiding a bias for reference genes with similar expression patterns (co-regulation). Based on these analyses we chose three reference genes, ACTB, UB2L3 and IF5A1 for further evaluation. Their consistency was assessed in an expression study of three known craniofacially expressed genes, sparc (or osteonectin), matrix metalloprotease 2 (mmp2) and sox9 (sex-determining region Y box 9 protein) using qPCR in embryo heads derived from four charr groups at three developmental time points. The three reference genes were found to be very suitable for studying expression differences between the morphotypes, enabling robust detection of small relative expression changes during charr development. Further, the results showed that sparc and mmp2 are differentially expressed in embryos of different Arctic charr morphotypes.Ehsan Pashay AhiJóhannes GuðbrandssonKalina H KapralovaSigríður R FranzdóttirSigurður S SnorrasonValerie H MaierZophonías O JónssonPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 6, p e66389 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ehsan Pashay Ahi Jóhannes Guðbrandsson Kalina H Kapralova Sigríður R Franzdóttir Sigurður S Snorrason Valerie H Maier Zophonías O Jónsson Validation of reference genes for expression studies during craniofacial development in arctic charr.
description	Arctic charr (Salvelinus alpinus) is a highly polymorphic species and in Lake Thingvallavatn, Iceland, four phenotypic morphs have evolved. These differences in morphology, especially in craniofacial structures are already apparent during embryonic development, indicating that genes important in the formation of the craniofacial features are expressed differentially between the morphs. In order to generate tools to examine these expression differences in Arctic charr, the aim of the present study was to identify reference genes for quantitative real-time PCR (qPCR). The specific aim was to select reference genes which are able to detect very small expression differences among different morphs. We selected twelve candidate reference genes from the literature, identified corresponding charr sequences using data derived from transcriptome sequencing (RNA-seq) and examined their expression using qPCR. Many of the candidate reference genes were found to be stably expressed, yet their quality-rank as reference genes varied considerably depending on the type of analysis used. In addition to commonly used software for reference gene validation, we used classical statistics to evaluate expression profiles avoiding a bias for reference genes with similar expression patterns (co-regulation). Based on these analyses we chose three reference genes, ACTB, UB2L3 and IF5A1 for further evaluation. Their consistency was assessed in an expression study of three known craniofacially expressed genes, sparc (or osteonectin), matrix metalloprotease 2 (mmp2) and sox9 (sex-determining region Y box 9 protein) using qPCR in embryo heads derived from four charr groups at three developmental time points. The three reference genes were found to be very suitable for studying expression differences between the morphotypes, enabling robust detection of small relative expression changes during charr development. Further, the results showed that sparc and mmp2 are differentially expressed in embryos of different Arctic charr morphotypes.
format	article
author	Ehsan Pashay Ahi Jóhannes Guðbrandsson Kalina H Kapralova Sigríður R Franzdóttir Sigurður S Snorrason Valerie H Maier Zophonías O Jónsson
author_facet	Ehsan Pashay Ahi Jóhannes Guðbrandsson Kalina H Kapralova Sigríður R Franzdóttir Sigurður S Snorrason Valerie H Maier Zophonías O Jónsson
author_sort	Ehsan Pashay Ahi
title	Validation of reference genes for expression studies during craniofacial development in arctic charr.
title_short	Validation of reference genes for expression studies during craniofacial development in arctic charr.
title_full	Validation of reference genes for expression studies during craniofacial development in arctic charr.
title_fullStr	Validation of reference genes for expression studies during craniofacial development in arctic charr.
title_full_unstemmed	Validation of reference genes for expression studies during craniofacial development in arctic charr.
title_sort	validation of reference genes for expression studies during craniofacial development in arctic charr.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/3016dd142811451294a002ebd90fcf9a
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Validation of reference genes for expression studies during craniofacial development in arctic charr.

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