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

Abstract

BackgroundThe 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.ResultsThe 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.ConclusionThe 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.

Long noncoding RNA are aberrantly expressed in vivo in the cystic fibrosis bronchial epithelium

“…As regards qRT-PCR, "traditional" reference genes such as GAPDH and ACTB have previously been found to be unsuitable for normalisation of mRNA levels in the airways of asthmatics (Glare et al, 2002), for gene expression studies employing alveolar macrophages from people with chronic obstructive pulmonary disease (Ishii et al, 2006b) and in bronchoalveolar cells from patients with other pulmonary diseases (Kriegova et al, 2008). A study to determine the reference genes with the most stable mRNA expression in bronchoalveolar cells from horses with inflammatory airway disease identified an optimal four gene reference panel (Beekman et al, 2011). Due to a lack of biological material at our disposal, we utilised a two gene normalisation approach using GAPDH and ACTB in combination, having validated that there is no significant difference in expression of either gene between bronchial epithelial cells subjects from CF or control groups; (iii) The sample sizes used were small; thus not allowing for high statistical power.…”

Peroxidase-Generated Apoplastic ROS Impair Cuticle Integrity and Contribute to DAMP-Elicited Defenses

“…These genes were tested to determine their stability across cDNA samples using the geNorm M -value principle in Genex 6 software (MultiD Analyses AB, Gothenburg, Sweden). The two most stable reference genes ( EF1α and UBQ10 ) had M -values < 0.3 across all samples and were therefore used for subsequent normalizations (Vandesompele et al, 2002; Beekman et al, 2011). All of the genes that were analyzed, along with their respective primers, are listed in Supplementary Table S1.…”

Evaluation of housekeeping genes for quantitative gene expression analysis in the equine kidney

“…It is assumed that reference genes, commonly known as housekeeping genes (HKGs), are stably expressed in the tissues/cells, so choosing a suitable internal reference gene is an important way of ensuring accurate interpretation of the results [11]. These HKGs do vary across tissues and organisms, so it is recommended that a combination of HKGs is used in order to acquire a much more stable and reliable reference [3]. …”

“…A number of studies on the appropriate internal controls for studying gene expression in equine tissues using RT-qPCR have been published [3, 4, 7, 9, 16, 21]. The aim of this study was to determine the most stably expressed HKGs in the equine kidney, which has not been studied yet, in order to use these for normalisation of gene expression in subsequent RT-qPCR experiments.…”

“…The difference in rankings may be explained by the different algorithms used and the different methodologies employed; where NormFinder identifies the single best gene with the most stable expression in the tissue, geNorm mostly detects the two (or more) optimal genes with the least variation in their expression ratio compared to other genes, but the results can be skewed by coregulation of genes in similar functional classes. NormFinder also takes the inter- and intra-group variations into account and is not significantly affected by coregulation of HKGs [3]. Most HKGs that were chosen in this study, apart from 18S and 28S , belong to different functional classes thus avoiding coregulation as a problem in the analysis.…”