Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with Inflammatory Airway Disease
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The best way to normalize quantitative real-time polymerase chain reaction (qRT-PCR) results is by using an internal control, or housekeeping gene. To date, no reference genes have been validated for expression studies of bronchoalveolar lavage cells of horses. The aim of this study was to determine a gene with a stable mRNA expression in bronchoalveolar lavage cells of horses with inflammatory Airway Disease (IAD) irrespective of treatment with intramuscular dexamethasone (DEX) or inhaled fluticasone propionate (FLUC). The mRNA expression of seven housekeeping genes (B2M, HPRT1, GAPDH, ACTB, UBB, RPL32 and SDHA) was investigated in bronchoalveolar lavage cells of seven horses with IAD. The horses were treated in a controlled randomized cross-over design study with DEX (seven horses) and FLUC (three horses). The seven housekeeping genes were tested with qRT-PCR to analyze the stability of the genes under the described circumstances. The results were analyzed with both the NormFinder software and the GeNorm software. These software’s rank the genes according to the stability of their expression. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) came out as the most stable housekeeping gene in the NormFinder software, under the described circumstances, with a stability factor of 0,013. GAPDH was followed by RPL32 (stability factor 0,025), HPRT (stability factor 0,027) and B2M (stability factor 0,028) in this order. SDHA (stability factor 0,033), ACTB (stability factor 0,034) and UBB (stability factor 0,040) completed the list with the highest stability factor. The best combination of two genes is GAPDH and RPL32 (stability factor 0,014). The GeNorm software ranks GAPDH and SDHA as the most stable housekeeping genes, followed by HPRT, RPL32, UBB and ACTB. The least stable expressed gene was B2M. Based on the pair-wise variation cut-off value (0,15), a combination of the four most stable housekeeping genes (GAPDH, SDHA, HPRT and RPL32) is accurate for normalization in this kind of studies. We thus recommend using GAPDH alone or in combination with either RPL32 or SDHA as housekeeping genes for gene expression studies in the BAL fluid of horses with IAD treated with steroids.