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.