Reference Genes for Real-Time Qpcr in Leukocytes from Asthmatic Patients Before and After Anti-Asthma Treatment

Gene 570 (2015) 71–77

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Research paper Reference genes for real-time qPCR in leukocytes from asthmatic patients before and after anti-asthma treatment

Carina E.P. Kozmus a,b,Uroš Potočnik a,c,⁎ a Centre for Human Molecular Genetics and Pharmacogenomics, Faculty of Medicine, University of Maribor, Taborska ulica 8, 2000 Maribor, Slovenia b Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alegre 823, 4150 Porto, Portugal c Laboratory for Biochemistry Molecular Biology and Genomics, Faculty of Chemistry and Chemical Engineering, University of Maribor, Smetanova ulica 17, 2000 Maribor, Slovenia article info abstract

Article history: The aim of this study was to develop a set of reference genes whose expression is stable and suitable for normal- Received 12 February 2015 ization of target gene expression measured in asthma patients during anti-asthmatic treatment. Received in revised form 27 May 2015 Real-time qPCR was used to determine expression of 7 candidate reference genes (18S rRNA, ACTB, B2M, GAPDH, Accepted 1 June 2015 POLR2A, RPL13A and RPL32) and 7 target genes in leukocytes from asthma patients before and after treatment Available online 4 June 2015 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 af- Keywords: ﬁ Inhaled corticosteroids fected the consistency of conclusions if the speci c investigated target gene is down-regulated or up-regulated Leukotriene receptor antagonist after anti-asthmatic therapy. Pharmagenetics The top-ranking reference genes determined by geNorm, when samples before and after therapy were analyzed Pharmagenomics (ACTB, B2M and GAPDH) were different from those (POLR2A and B2M) when only samples before treatment were Gene expression analyzed. Using only a single reference gene for normalization of 7 target gene expression compared to our strat- Housekeeping genes egy, 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. © 2015 Elsevier B.V. All rights reserved.

1. Introduction factors, but also interactions between the two (Hunter, 2005). Fam- ily studies indicated heritability of the disease as relatives of indi- It is well established that asthma is a complex disease, which viduals with asthma are at higher risk of asthma than individuals typically involves not only distinct genetic and environmental in the general population (Sandford et al., 1996). Therefore, in re- cent decades a lot of effort has been made to bring to light the genet-

Abbreviations: 18S rRNA, 18S ribosomal RNA gene; ACTB, β-Actin gene; ATS, American ics of asthma by determining which genes play a part in the disease. Thoracic Society; B2M, beta-2-microglobulin gene; Cq, quantification cycle; DAP, The ultimate goal of finding asthma associated genes is to provide D'Agostino-Pearson test; EDTA, ethylenediaminetetraacetic acid; EPHX2, cytoplasmic ep- insight into their complex regulatory networks, defining the path- oxide hydrolase 2 gene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase gene; ways involved in asthma in order to find targets for new treatments GSDMB, gasdermin B gene; ICS, inhaled corticosteroids; IQR, interquartile range; KS, (Barnes, 2010). Furthermore, such genes may assist the early detec- Kolmogorov–Smirnov test; LTRA, leukotriene receptor antagonist; M, expression stability measure; MAP3K2, mitogen-activated protein kinase kinase kinase 2 gene; MIQE, mini- tion, positive prognosis, and appropriate treatment of the disease. mum information for publication of quantitative real-time PCR experiments; NAEPP, Currently, asthma treatment is aimed at achieving and maintaining National Asthma Education and Prevention Program; NF, normalization factors; control of the clinical manifestations of the disease for prolonged ORMDL3, ORMDL sphingolipid biosynthesis regulator 3 gene; POLR2A, polymerase (RNA) periods. There are three main treatment options for controlling II (DNA directed) polypeptide A gene; PSMD3, proteasome (prosome, macropain) 26S sub- unit, non-ATPase, 3 gene; qPCR, quantitative Polymerase Chain Reaction; RPL13A, ribo- asthma, which may be combined: 1) corticosteroids 2) long acting somal protein L13a gene; RPL32,ribosomalproteinL32gene;SLC22A5, solute carrier beta2-agonists and 3) leukotriene inhibitors. However, patients family 22 (organic cation/carnitine transporter), member 5; SW, Shapiro–Wilk test; show substantial inter-individual variability in response to these TRIM35, tripartite motif-containing 35 gene; V, pairwise variation. medications and some even fail to manifest a response (Drazen ⁎ Corresponding author at: Centre for Human Molecular Genetics and Pharmacogenomics, et al., 2000). Pharmacogenetics and pharmacogenomics studies have Faculty of Medicine, University of Maribor, Taborska ulica 8, 2000 Maribor, Slovenia. E-mail addresses: [email protected] (C.E.P. Kozmus), already associated a number of genes to anti-asthmatic treatment out- [email protected] (U. Potočnik). come using genotype data (i.e. single nucleotide polymorphisms and

http://dx.doi.org/10.1016/j.gene.2015.06.001 0378-1119/© 2015 Elsevier B.V. All rights reserved. 72 C.E.P. Kozmus, U. Potočnik / Gene 570 (2015) 71–77 copy number variation). Recently, however, genomic studies have 2. Methods begun to focus on expression of genes involved in treatment out- come as potentially important biomarkers (Tse et al., 2011). 2.1. Study subjects The most common and powerful approach to individual gene expression analysis is real-time quantitative Polymerase Chain Reac- Asthmatic (allergic and non-allergic) patients, children aged be- tion (qPCR). With this technique it is necessary to normalize gene tween 7 and 18 years with mild or moderate persistent asthma treated expression data of the target gene with the gene expression data after 01.01.2008 in the University Medical Centre Maribor were enrolled of one or more reference genes from the same sample (Huggett in the study, considering inclusion and exclusion criteria (Perin et al., et al., 2005; Vandesompele et al., 2002). Normalization to reference 2011). Asthma was diagnosed according to NAEPP (National Asthma genes ensures that changes in target gene expression are selective Education and Prevention Program) and ATS (American Thoracic Society) and not reflective of variations in extraction yield, reverse transcrip- criteria. All patients with other chronic inflammatory diseases were ex- tion yield, and efficiency of amplification (Dheda et al., 2005). However, cluded from the study. Patients were free from any acute diseases or asth- the identification of a reference gene whose mRNA copy number per ma exacerbation at the time the blood samples were taken. Parents cell remains constant must be experimentally validated for particu- signed informed consent for children younger than 15 years old, while lar tissues or cell types and specific experimental designs because older children gave informed consent themselves. the expression of all genes is regulated during the cell cycle to con- Blood samples were collected during clinic hours from 78 asth- trol diverse cellular functions (Huggett et al., 2005; Vandesompele matic patients before treatment with any anti-asthma medication et al., 2002). (‘Pre-treatment’ group) with matching samples being obtained The use of β-actin (ACTB), glyceraldehyde-3-phosphate dehydroge- 2 years after treatment with ICS (n = 26) or LTRA (n = 38) (‘Post- nase (GAPDH) or 18S ribosomal RNA (18S rRNA) as reference genes is treatment’ group) (Table 1). In the ICS treatment group 200 μgof very common. However, several studies have shown that the expres- fluticasone dry powder (Flixotide diskus®, GSK Pharmaceuticals sion levels of classical reference genes can vary extensively in different S.A., Poland) per day was prescribed for children younger than tissues, different cell types and different disease stages and are therefore 12 years of age and 400 μg daily for older children. In the LTRA treat- unsuitable for normalization purposes (Vandesompele et al., 2002; Bas ment group patients were treated with montelukast — 5mgtablets et al., 2004; Li et al., 2011; Glare et al., 2002; Rubie et al., 2005). In were prescribed for children under 12 years old and 10 mg tablets spite of these observations, many published studies fail to mention a for older children. Non-atopic, non-asthmatic subjects were used validation process for the use of a particular reference gene for normal- as a control group (n = 71). ization. The normalization of data using a non-validated reference gene could lead to the loss of detection of small differences between expres- 2.2. RNA extraction sion of the genes of interest or to other inaccurate results and therefore erroneous conclusions (Huggett et al., 2005; Bas et al., 2004; Tricarico A 12 mL blood sample was drawn from each subject into tubes with et al., 2002). ethylenediaminetetraacetic acid (EDTA) for genetic analysis and proc- To the best of our knowledge no study has been performed to essed the same day. Total blood leukocytes were isolated using Ficoll- date to validate reference genes to normalize gene expression data Paque Plus (GE Healthcare, Sweden) gradient centrifugation, ac- from leukocytes in studies with asthmatic patients, and in particu- cording to the manufacturer's instructions. Total RNA was isolated lar, taking anti-asthma treatment into consideration. Analyses of using QIAzol® Lysis Reagent (QIAgen, USA). RNA concentrations gene expression from leukocytes of asthma patients are common ranged from ~0.1–1.17 μg/μL as determined by an ND1000 spectro- because they are easier to collect than specimens from the lower air- photometer and NanoDrop 3.0.1 software (NanoDrop Technologies, ways and share most abnormalities with T cells in bronchial mucosa Germany); 260/280 ratios ranged from 1.7 to 2.0. The integrity of RNA (Gladkevich et al., 2005). Therefore, the goal of this study was to samples was analyzed by electrophoresis on a 2% agarose gel and the identify the most suitable set of reference genes to be used to nor- 28S RNA was approximately twice as intense as the 18S rRNA (2:1 malize gene expression data in studies with asthmatic patients. ratio). All samples were immediately frozen and stored at −80 °C. We analyzed a panel of 7 candidate reference genes in isolated total blood leukocytes of a limited but representative number of 2.3. cDNA synthesis asthmatic patients before and after anti-asthma treatment with inhaled corticosteroids (ICS) or leukotriene receptor antagonist (LTRA), and First-strand cDNA was generated by reverse transcription of 1 μg control subjects. The selected candidate reference genes included 18S total RNA per sample with random primers and MultiScribe™ Reverse rRNA, ACTB, beta-2-microglobulin (B2M), GAPDH,polymerase(RNA)II Transcriptase (50 U/reaction) using High-Capacity cDNA Reverse Tran- (DNA directed) polypeptide A (POLR2A), ribosomal protein L13a scription kit (Cat. #4368813, Applied Biosystems, USA) in a final reac- (RPL13A)andribosomalproteinL32(RPL32). These are commonly tion volume of 20 μL. The first step of reverse transcriptase reaction used as endogenous controls and good candidates for normalization of was performed at 25 °C for 10 min followed by a 2 h incubation period gene expression data for pharmacogenetic studies that were report- ed to be among the most stable reference genes in a wide variety of Table 1 studies (Vandesompele et al., 2002; Bas et al., 2004; Valceckiene Subjects profile. et al., 2010; Mane et al., 2008; Zhang et al., 2005; Saviozzi et al., Asthmatics Controls 2006; Wang et al., 2012; Potashnikova et al., 2015; Ledderose N7871 et al., 2011). We also evaluated the impact of different normalization Age, mean ± SD 11.59 ± 2.70 21.59 ± 2.90 strategies in the relative change of expression of 7 target genes located Sex, n male/female 42/36 33/38 in asthma associated loci, which included ORMDL sphingolipid biosynthe- Allergic, n (%) 55 (70.5%) – sis regulator 3 (ORMDL3), proteasome (prosome, macropain) 26S sub- Non-allergic, n (%) 21 (26.9%) – – unit, non-ATPase, 3 (PSMD3), gasdermin B (GSDMB), mitogen-activated Unknown atopy, n (%) 2 (2.6%) ICSa, n (%) 26 (33.3%) – protein kinase kinase kinase 2 (MAP3K2), solute carrier family 22 (organic LTRAb, n (%) 38 (48.7) – cation/carnitine transporter), member 5 (SLC22A5), tripartite motif- ICS — Inhaled corticosteroids; LTRA — Leukotriene receptor antagonist; SD — Standard containing 35 (TRIM35) and epoxide hydrolase 2, cytoplasmic (EPHX2) deviation. fl a in the samples as a preliminary study of the in uence of treatment on Patients who agreed with second blood withdrawal after 2 years of ICS treatment. theexpressionofthesegenes. b Patients who agreed with second blood withdrawal after 2 years of LTRA treatment. 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