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Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model

Preterm newborns often require invasive support, however even brief periods of supported ventilation applied inappropriately to the lung can cause injury. Real-time quantitative reverse transcriptase-PCR (qPCR) has been extensively employed in studies of ventilation-induced lung injury with the refe...

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Autores principales: Pereira-Fantini, Prue M., Rajapaksa, Anushi E., Oakley, Regina, Tingay, David G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4876477/
https://www.ncbi.nlm.nih.gov/pubmed/27210246
http://dx.doi.org/10.1038/srep26476
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author Pereira-Fantini, Prue M.
Rajapaksa, Anushi E.
Oakley, Regina
Tingay, David G.
author_facet Pereira-Fantini, Prue M.
Rajapaksa, Anushi E.
Oakley, Regina
Tingay, David G.
author_sort Pereira-Fantini, Prue M.
collection PubMed
description Preterm newborns often require invasive support, however even brief periods of supported ventilation applied inappropriately to the lung can cause injury. Real-time quantitative reverse transcriptase-PCR (qPCR) has been extensively employed in studies of ventilation-induced lung injury with the reference gene 18S ribosomal RNA (18S RNA) most commonly employed as the internal control reference gene. Whilst the results of these studies depend on the stability of the reference gene employed, the use of 18S RNA has not been validated. In this study the expression profile of five candidate reference genes (18S RNA, ACTB, GAPDH, TOP1 and RPS29) in two geographical locations, was evaluated by dedicated algorithms, including geNorm, Normfinder, Bestkeeper and ΔCt method and the overall stability of these candidate genes determined (RefFinder). Secondary studies examined the influence of reference gene choice on the relative expression of two well-validated lung injury markers; EGR1 and IL1B. In the setting of the preterm lamb model of lung injury, RPS29 reference gene expression was influenced by tissue location; however we determined that individual ventilation strategies influence reference gene stability. Whilst 18S RNA is the most commonly employed reference gene in preterm lamb lung studies, our results suggest that GAPDH is a more suitable candidate.
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spelling pubmed-48764772016-06-06 Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model Pereira-Fantini, Prue M. Rajapaksa, Anushi E. Oakley, Regina Tingay, David G. Sci Rep Article Preterm newborns often require invasive support, however even brief periods of supported ventilation applied inappropriately to the lung can cause injury. Real-time quantitative reverse transcriptase-PCR (qPCR) has been extensively employed in studies of ventilation-induced lung injury with the reference gene 18S ribosomal RNA (18S RNA) most commonly employed as the internal control reference gene. Whilst the results of these studies depend on the stability of the reference gene employed, the use of 18S RNA has not been validated. In this study the expression profile of five candidate reference genes (18S RNA, ACTB, GAPDH, TOP1 and RPS29) in two geographical locations, was evaluated by dedicated algorithms, including geNorm, Normfinder, Bestkeeper and ΔCt method and the overall stability of these candidate genes determined (RefFinder). Secondary studies examined the influence of reference gene choice on the relative expression of two well-validated lung injury markers; EGR1 and IL1B. In the setting of the preterm lamb model of lung injury, RPS29 reference gene expression was influenced by tissue location; however we determined that individual ventilation strategies influence reference gene stability. Whilst 18S RNA is the most commonly employed reference gene in preterm lamb lung studies, our results suggest that GAPDH is a more suitable candidate. Nature Publishing Group 2016-05-23 /pmc/articles/PMC4876477/ /pubmed/27210246 http://dx.doi.org/10.1038/srep26476 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Pereira-Fantini, Prue M.
Rajapaksa, Anushi E.
Oakley, Regina
Tingay, David G.
Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model
title Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model
title_full Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model
title_fullStr Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model
title_full_unstemmed Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model
title_short Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model
title_sort selection of reference genes for gene expression studies related to lung injury in a preterm lamb model
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4876477/
https://www.ncbi.nlm.nih.gov/pubmed/27210246
http://dx.doi.org/10.1038/srep26476
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