Abstract
Reference genes are essential for studying mRNA expression with quantitative PCR (qPCR). We investigated 11 potential neutrophil reference genes (RPL19, GAPDH, ACTB, B2M, HPRT, G6PD, TFRC, PGK1, YWHAZ, SDHA and GYPC) for sheep under disease conditions of foot rot (FR) and with or without Se supplementation. Initial screening was based on gene expression level (<28 Cq cycles) and variability (SD < 1.5 Cq cycles) and excluded TFRC, GYPC and HPRT from further analysis. Expression stability of the remaining genes was evaluated using four software programs: geNorm, NormFinder, BestKeeper and the comparative delta Cq method. The neutrophil reference genes, G6PD, YWHAZ, GAPDH, RPL19 and SDHA, consistently ranked among the top five most stable genes under these experimental conditions. The SDHA gene expression was not stable in FR-diseased sheep receiving Se treatment and, thus, cannot be recommended as a reference gene. The commonly used genes, PGK1, ACTB and B2M, were not reliable reference genes, underscoring the need to validate neutrophil reference genes under different experimental conditions. Multiple references genes rather than a single gene may provide more robust and reliable results. The best pair of reference genes was SDHA/G6PD in healthy sheep and GADPH/YWHAZ in FR-diseased sheep.
Highlights
Quantitative PCR is a powerful tool for gene expression analysis [1]
Expression Level of Neutrophil Reference Genes Evaluated in This Study
The most variation was associated with HPRT (24.52 ± 2.22; Cq range, 6.12), which was the only gene that did not pass the test for normal distribution (Shapiro-Wilk W = 0.79; p = 0.02), and HPRT was excluded from further analysis
Summary
Quantitative PCR (qPCR) is a powerful tool for gene expression analysis [1]. Expression data from genes of interest are normalized against reference genes to correct for the initial amount of starting material in order to determine expression differences with disease or in response to treatment.Reference genes, which are often referred to as housekeeping genes, are assumed to be constitutively expressed; reference gene expression may vary depending upon the cell type analyzed and experimental conditions [2,3,4,5]. Expression data from genes of interest are normalized against reference genes to correct for the initial amount of starting material in order to determine expression differences with disease or in response to treatment. Prior to examining expression data from neutrophils in healthy and foot rot (FR) affected sheep, the selection of appropriate reference genes is critical. We have previously reported that sheep affected with FR have lower whole blood selenium (Se) concentrations and that Se supplementation in conjunction with routine control practices accelerates recovery from FR [7]. In the previously reported study, sheep affected with FR were injected monthly for 15 months with either Se or saline, whereas healthy sheep received no treatment [7]. Before assessing the relative abundance of mRNA for genes associated with neutrophil functions, neutrophil reference genes in healthy and FR-diseased sheep, with and without Se supplementation, are needed
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