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BMC molecular biology
Jan 28, 2011;12 5.

Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease.

Laura Beekman, Triin Tohver, Rkia Dardari, Renaud Léguillette
Author Information
  1. Laura Beekman: Departement of Veterinary Clinical and Diagnostic Sciences, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada.
PMID: 21272375 DOI: 10.1186/1471-2199-12-5

Abstract

BACKGROUND: The stability of reference genes has a tremendous effect on the results of relative quantification of genes expression by quantitative polymerase chain reaction. Equine Inflammatory Airway Disease (IAD) is a common condition often treated with corticosteroids. The diagnosis of IAD is based on clinical signs and bronchoalveolar lavage (BAL) fluid cytology. The aim of this study was to identify reference genes with the most stable mRNA expression in the BAL cells of horses with IAD irrespective of corticosteroids treatment.
RESULTS: The expression stability of seven candidate reference genes (B2M, HPRT, GAPDH, ACTB, UBB, RPL32, SDHA) was determined by qRT-PCR in BAL samples taken pre- and post- treatment with dexamethasone and fluticasone propionate for two weeks in 7 horses with IAD. Primers' efficiencies were calculated using LinRegPCR. NormFinder, GeNorm and qBasePlus softwares were used to rank the genes according to their stability. GeNorm was also used to determine both the ideal number and the best combination of reference genes. GAPDH was found to be the most stably expressed gene with the three softwares. GeNorm ranked B2M as the least stable gene. Based on the pair-wise variation cut-off value determined with GeNorm, the number of genes required for optimal normalization was four and included GAPDH, SDHA, HPRT and RPL32.
CONCLUSION: The geometric mean of GAPDH, HPRT, SDHA and RPL32 is recommended for accurate normalization of quantitative PCR data in BAL cells of horses with IAD treated with corticosteroids. If only one reference gene can be used, then GAPDH is recommended.

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MeSH Term

Animals
Bronchoalveolar Lavage
Bronchoalveolar Lavage Fluid
Female
Gene Expression Profiling
Horse Diseases
Horses
Inflammation
Lung Diseases
Male
Reference Standards
Reverse Transcriptase Polymerase Chain Reaction
Journal Article Research Support, Non-U.S. Gov't Validation Study

Word Cloud

Created with Highcharts 10.0.0genesreferenceIADGAPDHexpressionBALhorsesGeNormgenestabilitycorticosteroidscellsHPRTRPL32SDHAusedquantitativetreatedbronchoalveolarlavagestabletreatmentB2MdeterminedsoftwaresnumbernormalizationrecommendedBACKGROUND:tremendouseffectresultsrelativequantificationpolymerasechainreactionEquineInflammatoryAirwayDiseasecommonconditionoftendiagnosisbasedclinicalsignsfluidcytologyaimstudyidentifymRNAirrespectiveRESULTS:sevencandidateACTBUBBqRT-PCRsamplestakenpre-post-dexamethasonefluticasonepropionatetwoweeks7Primers'efficienciescalculatedusingLinRegPCRNormFinderqBasePlusrankaccordingalsodetermineidealbestcombinationfoundstablyexpressedthreerankedleastBasedpair-wisevariationcut-offvaluerequiredoptimalfourincludedCONCLUSION:geometricmeanaccuratePCRdataonecanEvaluationsuitablestudiesinflammatoryairwaydisease

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