title: Reference genes for real-time qPCR in leukocytes from asthmatic patients before and after anti-asthma treatment.
abstract: The aim of this study was to develop a set of reference genes whose expression is stable and suitable for normalization of target gene expression measured in asthma patients during anti-asthmatic treatment. Real-time qPCR was used to determine expression of 7 candidate reference genes (18S rRNA, ACTB, B2M, GAPDH, POLR2A, RPL13A and RPL32) and 7 target genes in leukocytes from asthma patients before and after treatment with inhaled corticosteroids and leukotriene receptor antagonist. Variance of Cq values was analyzed and stability ranking was determined with geNorm. We further investigated how the different normalization strategies affected the consistency of conclusions if the specific investigated target gene is down-regulated or up-regulated after anti-asthmatic therapy. The top-ranking reference genes determined by geNorm, when samples before and after therapy were analyzed (ACTB, B2M and GAPDH) were different from those (POLR2A and B2M) when only samples before treatment were analyzed. Using only a single reference gene for normalization of 7 target gene expression compared to our strategy, there would be as low as 19% of consistency in conclusions. We suggest the use of the geometric mean of ACTB, B2M and GAPDH for normalization of qPCR data of target genes in pharmacogenomics studies in asthma patients before and after anti-asthmatic therapy, however if gene expression is measured only before anti-asthmatic treatment, we recommend the use of the geometric mean of POLR2A and B2M.
authors.affiliation: Centre for Human Molecular Genetics and Pharmacogenomics, Faculty of Medicine, University of Maribor, Taborska ulica 8, 2000 Maribor, Slovenia; Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alegre 823, 4150 Porto, Portugal. Electronic address: carina.kozmus@gmail.com.
authors.forename: Carina E P
authors.initials: CE
authors.lastname: Kozmus
authors.affiliation: Centre for Human Molecular Genetics and Pharmacogenomics, Faculty of Medicine, University of Maribor, Taborska ulica 8, 2000 Maribor, Slovenia; Laboratory for Biochemistry Molecular Biology and Genomics, Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova ulica 17, 2000 Maribor, Slovenia. Electronic address: uros.potocnik@uni-mb.si.
authors.forename: Uroš
authors.initials: U
authors.lastname: Potočnik
mesh_terms: D000293:Adolescent; D000730:Androstadienes / Q000494:pharmacology* / Q000627:therapeutic use; D018927:Anti-Asthmatic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001249:Asthma / Q000097:blood* / Q000188:drug therapy; D016022:Case-Control Studies; D002648:Child; D005260:Female; D000068298:Fluticasone; D020869:Gene Expression Profiling / Q000592:standards*; D020043:Genes, Essential; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D059467:Transcriptome; D055815:Young Adult
chemical_list: D000730:Androstadienes; D018927:Anti-Asthmatic Agents; D000068298:Fluticasone
keywords: Gene expression; Housekeeping genes; Inhaled corticosteroids; Leukotriene receptor antagonist; Pharmagenetics; Pharmagenomics