Abstract
Reference genes are essential for studying mRNA expression with quantitative PCR (qPCR). We investigated 11 candidate whole-blood neutrophil reference genes (ACTB, B2M, G6PD, GAPDH, GYPC, HPRT, PGK1, RPL19, SDHA, TFRC, and YWHAZ) for beef calves, both males and females, with or without selenium supplementation. Initial screening was based on gene expression level (<28 Cq cycles), variability (SD < 1.5 Cq cycles), excluded GYPC and TFRC 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, YWHAZ, PGK1, and RPL19, consistently ranked among the top four most stable genes under these experimental conditions. The commonly used reference genes, ACTB and HPRT, were not reliable, underscoring the need to validate neutrophil reference genes under different experimental conditions. Multiple reference genes rather than a single gene may provide more robust and reliable results. The best pair of reference genes in whole-blood neutrophils from beef calves overall was PGK1|YWHAZ.
Highlights
Quantitative PCR is one of the most common techniques used for evaluating gene expression [1]
We investigated 11 candidate whole-blood neutrophil reference genes (ACTB, B2M, glucose-6phosphate dehydrogenase (G6PD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycophorin C (GYPC), hypoxanthine phosphoribosyl-transferase 1 (HPRT), phosphoglycerate kinase 1 (PGK1), ribosomal protein L19 (RPL19), SDHA, transferrin receptor (TFRC), and YWHAZ) for beef calves, both males and females, with or without selenium supplementation
We found that SDHA and glucose-6phosphate dehydrogenase (G6PD) in healthy sheep, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Summary
Quantitative PCR (qPCR) is one of the most common techniques used for evaluating gene expression [1]. Neutrophils are the most numerous and important cellular component of innate immunity They serve as the body’s first line of defense against invading microorganisms. In one study with human neutrophils, suitable reference genes included a TATA box binding protein (TBP), beta-actin (ACTB), and succinate dehydrogenase complex subunit A (SDHA) [11]. In another human study, guanine nucleotide binding protein, ß-peptide 2-like (GNB2L1), hypoxanthine phosphoribosyl-transferase 1 (HPRT), ribosomal protein L32 (RPL32), ACTB, and beta-2-microglobulin (B2M) were suggested as suitable reference genes in gene expression studies of neutrophils [10]. We found that SDHA and glucose-6phosphate dehydrogenase (G6PD) in healthy sheep, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
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