Abstract
BackgroundTo 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.ResultsThe 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.ConclusionsThere 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.
Highlights
To obtain reliable quantitative real-time PCR data, normalization relative to stable housekeeping genes (HKGs) is required
It is crucial to know whether the expression stability of HKGs in alveolar macrophages (AMs) is affected by various pathogen-associated molecular patterns (PAMPs) from infectious agents but these data are currently unavailable for pigs
Due to the new influx of data suggesting alterations in mRNA expression according to bacteria type, we feel that beside therapy uses or experimental condition, there needs to be special consideration given to the selection of HKGs based upon the bacterial pathogen identification
Summary
To obtain reliable quantitative real-time PCR data, normalization relative to stable housekeeping genes (HKGs) is required. Three different algorithms (geNorm, Normfinder and BestKeeper) were applied to assess the stability of HKGs. 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 bacterial lung diseases. 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
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