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Reference Genes for Quantitative Gene Expression Studies in Multiple Avian Species

Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian s...

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Autores principales: Olias, Philipp, Adam, Iris, Meyer, Anne, Scharff, Constance, Gruber, Achim D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057121/
https://www.ncbi.nlm.nih.gov/pubmed/24926893
http://dx.doi.org/10.1371/journal.pone.0099678
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author Olias, Philipp
Adam, Iris
Meyer, Anne
Scharff, Constance
Gruber, Achim D.
author_facet Olias, Philipp
Adam, Iris
Meyer, Anne
Scharff, Constance
Gruber, Achim D.
author_sort Olias, Philipp
collection PubMed
description Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species.
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spelling pubmed-40571212014-06-18 Reference Genes for Quantitative Gene Expression Studies in Multiple Avian Species Olias, Philipp Adam, Iris Meyer, Anne Scharff, Constance Gruber, Achim D. PLoS One Research Article Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species. Public Library of Science 2014-06-13 /pmc/articles/PMC4057121/ /pubmed/24926893 http://dx.doi.org/10.1371/journal.pone.0099678 Text en © 2014 Olias et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Olias, Philipp
Adam, Iris
Meyer, Anne
Scharff, Constance
Gruber, Achim D.
Reference Genes for Quantitative Gene Expression Studies in Multiple Avian Species
title Reference Genes for Quantitative Gene Expression Studies in Multiple Avian Species
title_full Reference Genes for Quantitative Gene Expression Studies in Multiple Avian Species
title_fullStr Reference Genes for Quantitative Gene Expression Studies in Multiple Avian Species
title_full_unstemmed Reference Genes for Quantitative Gene Expression Studies in Multiple Avian Species
title_short Reference Genes for Quantitative Gene Expression Studies in Multiple Avian Species
title_sort reference genes for quantitative gene expression studies in multiple avian species
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057121/
https://www.ncbi.nlm.nih.gov/pubmed/24926893
http://dx.doi.org/10.1371/journal.pone.0099678
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