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

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    18
    Author
    Pereira-Fantini, PM; Rajapaksa, AE; Oakley, R; Tingay, DG
    Date
    2016-05-23
    Source Title
    Scientific Reports
    Publisher
    NATURE PUBLISHING GROUP
    University of Melbourne Author/s
    Tingay, David; Rajapaksa, Anushi; Pereira-Fantini, Prudence
    Affiliation
    Paediatrics (RCH)
    Metadata
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    Document Type
    Journal Article
    Citations
    Pereira-Fantini, P. M., Rajapaksa, A. E., Oakley, R. & Tingay, D. G. (2016). Selection of Reference Genes for Gene Expression Studies related to lung injury in a preterm lamb model. SCIENTIFIC REPORTS, 6 (1), https://doi.org/10.1038/srep26476.
    Access Status
    Open Access
    URI
    http://hdl.handle.net/11343/259318
    DOI
    10.1038/srep26476
    Abstract
    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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