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. 2011 Jan 28:12:5.
doi: 10.1186/1471-2199-12-5.

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

Laura Beekman  1 , Triin Tohver, Rkia Dardari, Renaud Léguillette
Affiliations

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

Laura Beekman et al. BMC Mol Biol. .

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.

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Figures

Figure 1
Figure 1
Bronchoalveolar lavage fluid differential cell counts in horses with IAD pre- and post- treatment with corticosteroids. DEX indicates dexamethasone. FLUC indicates fluticasone. Bars indicate standard deviation. The figure shows the differential cell counts for neutrophils, lymphocytes, macrophages, mast cells and eosinophils as a percentage of the total amount of nucleated cells. * indicates a significant difference between pre- and post- treatment (p < 0.05).
Figure 2
Figure 2
Average cycle threshold (Ct) values of candidate reference genes tested in the bronchoalveolar lavage fluid of horses with IAD. The values are RT-PCR cycle threshold numbers (Ct values) pre-and post-treatment in horses with IAD treated with corticosteroids. The bars indicate standard deviation. Letters indicate a significant difference in average Ct value. Average Ct values that have the same letter are not significantly different.
Figure 3
Figure 3
Ranking of seven candidate reference genes using NormFinder and GeNorm softwares. (A) NormFinder ranks the genes based on a calculated stability value. The lower the stability value, the higher the expression stability. (B) GeNorm ranks the candidate reference genes based on their stability parameter M. The lower the M value, the higher the expression stability.
Figure 4
Figure 4
Determination of the optimal number of reference genes for normalization. The GeNorm software calculates the normalization factor from an increasing number of genes (starting with at least two) for which the variable V defines the pairwise variation between two sequential normalization factors. The lower the pairwise variation, the better is the combination of genes for reference. V4/5 for example, shows the variation between the normalization factors of four genes in relation to five genes and shows that four genes is the combination providing the lowest pairwise variation.
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