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dc.contributor.authorSandercock DAen
dc.contributor.authorCoe JEen
dc.contributor.authorDi Giminiani Pen
dc.contributor.authorEdwards SAen
dc.date.accessioned2017-10-11T10:53:24Z
dc.date.available2017-10-11T10:53:24Z
dc.date.issued2017
dc.identifier.citation114en
dc.identifier.issn0034-5288
dc.identifier.urihttps://doi.org/10.1016/j.rvsc.2017.09.025
dc.identifier.urihttp://hdl.handle.net/11262/11318
dc.description.abstractRNA expression levels for genes of interest must be normalised with appropriate reference or “housekeeping” genes that are stably expressed across samples and treatments. This study determined the most stable reference genes from a panel of 6 porcine candidate genes: beta actin (ACTB), beta-2-microglobulin (B2M), eukaryotic elongation factor 1 gamma-like protein (eEF-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), Ubiquitin C (UBC) in sacral dorsal root ganglia and spinal cord samples collected from 16 tail docked pigs (2/3rds of tail amputated) 1, 4, 8 and 16 weeks after tail injury (4 pigs/time point). Total RNA from pooled samples was measured by SYBRgreen real-time quantitative PCR. Cycle threshold values were analysed using geNorm, BestKeeper and NormFinder PCR analysis software. Average expression stability and pairwise variation values were calculated for each candidate reference gene. GeNorm analysis identified the most stable genes for normalisation of gene expression data to be GAPDH > eEF-1 > UBC > B2M > ACTB > SDHA for dorsal root ganglia and ACTB > SDHA > UBC > B2M > GAPDH > eEF-1 for spinal cord samples. Expression stability estimates were verified by BestKeeper and NormFinder analysis. Expression stability varied between genes within and between tissues. Validation of most stably expressed reference genes was performed by normalisation of calcitonin gene related polypeptide beta (CALCB). The results show similar patterns of CALCB expression when the best reference genes selected by all three programs were used. GAPDH, eEF-1 and UBC are suitable reference genes for porcine dorsal root ganglia samples, whereas ACTB, SDHA and UBC are more appropriate for spinal cord samples.en
dc.description.sponsorshipBBSRC - BB/L013584/1 in support of the ANIWHA Era-Net initiative (FareWellDock Project)en
dc.language.isoen_USen
dc.relation.isformatof14696en
dc.relation.ispartofResearch in Veterinary Scienceen
dc.rights© 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectPigen
dc.subjectRT-qPCRen
dc.subjectDorsal root gangliaen
dc.subjectSpinal corden
dc.subjectReference genesen
dc.subjectNormalisationen
dc.titleDetermination of stable reference genes for RT-qPCR expression data in mechanistic pain studies on pig dorsal root ganglia and spinal corden
dc.typeArticleen
dc.description.versionVersion of record
dc.extent.pageNumbers493-501en
rioxxterms.publicationdate2017-09-28
rioxxterms.typeJournal Article/Reviewen
dcterms.dateAccepted2017-09-27
refterms.accessExceptionNAen
refterms.dateDeposit2017-10-11
refterms.depositExceptionpublishedGoldOAen
refterms.panelUnspecifieden
refterms.technicalExceptionNAen
refterms.versionVoRen


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© 2017 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY license
Except where otherwise noted, this item's license is described as © 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license