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With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies
Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science. (Ctr Ecol & Evolut Microbial Model Syst)ORCID iD: 0000-0003-2849-1094
Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.ORCID iD: 0000-0002-1152-4235
2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 11, e0141853Article, review/survey (Refereed) Published
Abstract [en]

The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with beta-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

Place, publisher, year, edition, pages
2015. Vol. 10, no 11, e0141853
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
URN: urn:nbn:se:lnu:diva-47712DOI: 10.1371/journal.pone.0141853ISI: 000364430700041PubMedID: 26555275OAI: oai:DiVA.org:lnu-47712DiVA: diva2:876723
Available from: 2015-12-04 Created: 2015-12-04 Last updated: 2016-12-15Bibliographically approved

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CiteExportLink to record
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Citation style
  • apa
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