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Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease.

ABSTRACT: BACKGROUND: The stability of reference genes has a tremendous effect on the results of relative quantification of genes expression by quantitative polymerase chain reaction. Equine Inflammatory Airway Disease (IAD) is a common condition often treated with corticosteroids. The diagnosis of IAD is based on clinical signs and bronchoalveolar lavage (BAL) fluid cytology. The aim of this study was to identify reference genes with the most stable mRNA expression in the BAL cells of horses with IAD irrespective of corticosteroids treatment. RESULTS: The expression stability of seven candidate reference genes (B2M, HPRT, GAPDH, ACTB, UBB, RPL32, SDHA) was determined by qRT-PCR in BAL samples taken pre- and post- treatment with dexamethasone and fluticasone propionate for two weeks in 7 horses with IAD. Primers' efficiencies were calculated using LinRegPCR. NormFinder, GeNorm and qBasePlus softwares were used to rank the genes according to their stability. GeNorm was also used to determine both the ideal number and the best combination of reference genes. GAPDH was found to be the most stably expressed gene with the three softwares. GeNorm ranked B2M as the least stable gene. Based on the pair-wise variation cut-off value determined with GeNorm, the number of genes required for optimal normalization was four and included GAPDH, SDHA, HPRT and RPL32. CONCLUSION: The geometric mean of GAPDH, HPRT, SDHA and RPL32 is recommended for accurate normalization of quantitative PCR data in BAL cells of horses with IAD treated with corticosteroids. If only one reference gene can be used, then GAPDH is recommended.

SUBMITTER: Beekman L

PROVIDER: S-EPMC3039571 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease.

Beekman Laura L Tohver Triin T Dardari Rkia R Léguillette Renaud R

BMC molecular biology 20110128

<h4>Background</h4>The stability of reference genes has a tremendous effect on the results of relative quantification of genes expression by quantitative polymerase chain reaction. Equine Inflammatory Airway Disease (IAD) is a common condition often treated with corticosteroids. The diagnosis of IAD is based on clinical signs and bronchoalveolar lavage (BAL) fluid cytology. The aim of this study was to identify reference genes with the most stable mRNA expression in the BAL cells of horses with ...[more]

PMID: 21272375

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Project description:BACKGROUND: As an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs. RESULTS: The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P?<?0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. 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. CONCLUSION: There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.

Dataset Information

Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease.

Publications

Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease.

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