Reference gene selection for quantitative PCR studies in bovine neutrophils  

作　　者：William R. Vorachek Gerd Bobe Jean A. Hall 

机构地区：[1]Department of Animal and Rangeland Sciences, College of Agricultural Sciences, Linus Pauling Institute, Oregon State University, Corvallis, USA [2]Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, USA

出　　处：《Advances in Bioscience and Biotechnology》2013年第10期6-14,共9页生命科学与技术进展（英文）

摘　　要：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.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.

关 键 词：Blood NEUTROPHILS BOS TAURUS qPCR REFERENCE Genes SELENIUM Treatment 

分 类 号：R73[医药卫生—肿瘤]