Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA

Cinar et al. BMC Research Notes 2012, 5:107 http://www.biomedcentral.com/1756-0500/5/107 RESEARCH ARTICLE Open Access Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA Mehmet Ulas Cinar1, Mohammad Ariful Islam1,2, Muhammad Jasim Uddin1,2, Ernst Tholen1, Dawit Tesfaye1, Christian Looft1 and Karl Schellander1* Abstract Background: To obtain reliable quantitative real-time PCR data, normalization relative to stable housekeeping genes (HKGs) is required. However, in practice, expression levels of ‘typical’ housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in pigs are relatively rare and have never been performed in porcine alveolar macrophages (AMs). In this study, expression stability of putative housekeeping genes were identified in the porcine AMs in response to the stimulation with two pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Three different algorithms (geNorm, Normfinder and BestKeeper) were applied to assess the stability of HKGs. Results: The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by qRT-PCR in AMs that were stimulated by LPS and LTA in vitro. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.0001). geNorm software revealed that SDHA, B2M and RPL4 showed a high expression stability in the irrespective to the stimulation group, while SDHA, YWHAZ and RPL4 showed high stability in non-stimulated control group. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that SDHA was the most stable gene in all the groups. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study. Conclusions: There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms. In conclusion, the geometric mean of the SDHA, YWHAZ and RPL4 seemed to be the most appropriate combination of HKGs for accurate normalization of gene expression data in porcine AMs without knowing the type of bacterial pathogenic status of the animals. Keywords: Candidate reference genes, Alveolar macrophage, LPS, LTA, Pigs Background Alveolar macrophages (AMs) are thought to be critical in the pathogenesis of several lung diseases [1]. Swine respiratory diseases, which has been described worldwide, affects swine of all ages and has a serious impact on economy, ecology and animal welfare in the pig rearing industry [2]. Both Gram-positive and Gram-negative * Correspondence: 1 Institute of Animal Sciences, Unit of Animal Breeding and Husbandry, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany Full list of author information is available at the end of the article bacteria are causing respiratory disease in pigs [3]. As an in vitro model for the development of lung inflammation, AMs stimulation with PAMPs in culture is being frequently used for immunogenetic research in pigs [4-7]. Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are the PAMPs of the Gram-negative and the Gram-positive bacterial cell wall that cause activation of an acute inflammatory response in vitro as well as in vivo. Gene expression assay is a common way to investigate the defensive role of AMs in the bacterial infections as well as to dissect the pathogenesis of © 2012 Cinar et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cinar et al. BMC Research Notes 2012, 5:107 http://www.biomedcentral.com/1756-0500/5/107 bacterial lung diseases. With this purposes, several studies focusing on gene expressions have been conducted in AMs in vitro [4-7]. The gene expression are required to normalize for housekeeping genes (HKGs) which have tremendous effect on the results of expression study [8]. Therefore, it is crucial to know whether the expression stability of HKGs in AMs is affected by various PAMPs from infectious agents but these data are currently unavailable for pigs. Quantitative real-time PCR (qRT-PCR) is a powerful technique for gene expression studies, which have become increasingly important in a large number of clinical and scientific fields [8,9]. Besides being a powerful technique, inappropriate data normalization is the most important problem in qRT-PCR [8]. For an exact comparison of mRNA transcription in different samples or tissues, it is crucial to choose the appropriate reference gene [9]. The most accepted approach to mRNA quantification is normalization of the expression level of a gene of interest (target gene) to the expression level of a stably expressed internal reference gene [8,9]. Normalizing to a reference gene is a widely used method because it is simple in theory. Normalizing to a single reference gene is often used but Vandesompele et al. [8] suggested that geometric mean of multiple carefully selected HKGs is recommendable and suitable for accurate normalization. The normalization adjusts for differences in the quality or quantity of template RNA or starting material and differences in RNA preparation and cDNA synthesis, since the reference gene is exposed to the same preparation steps as the gene of interest. This allows the direct comparison of normalized transcript expression levels between samples. Reference genes should ideally be constitutively expressed by all cell types and should not be affected by disease and experimental procedure. To date, a universal reference gene has not been identified. HKGs are most commonly used reference genes [8]. Although HKGs are expressed by any cell, their expression varies among different cell types/organs, age, sex and treatment or experimental conditions [10-17]. Use of HKGs as reference genes for a particular sample type should be, therefore, validated. Ideally, the conditions of the experiment should not influence the expression of the reference genes [18]. However, the mRNA expression of reference genes from different cells and tissues [18-21] such as from AMs [1,10] may fluctuate due to infectious agents in vitro. Alveolar macrophages are being used as an important model to dissect the pathogenesis and genetics behind the infection through gene expression studies [5,6,22,23]. To date, no reference genes have been validated for expression studies of AMs in pigs. The aim of this study was therefore to identify a set of stably expressed reference genes in porcine AMs cells Page 2 of 14 irrespective of stimulation as (...truncated)