Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils

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.

Reference gene selection for quantitative PCR studies in bovine neutrophils

“…More reliable qPCR data normalization should be achieved using a combination of genes from different biological pathways, rather than a single gene. Our selection of reference genes for bovine neutrophils differs from results obtained for human neutrophils whereby recommended reference genes included TBP, ACTB, and SDHA [11] or GNB2L1, HPRT1, RPL32, ACTB, and B2M [10], and also differs from results we obtained for sheep neutrophils, whereby SDHA|G6PD and GAPDH|YWHAZ were the best pairs of references genes [13]. In lactating dairy cows, YWHAZ along with SDHA and 18S rRNA, were considered the most suitable reference genes for polymorphonuclear leukocytes [14].…”

“…Reference genes were selected based upon a literature review to find genes that covered a wide variety of functions and pathways: ACTB, GAPDH, and B2M [19]; HPRT, SDHA, and YWHAZ [14], RPL19 [25], and four commonly used ovine neutrophil reference genes (G6PD, TFRC, PGK1, and GYPC) [12,13]. All primers were synthesized by Sigma Aldrich (St. Louis, MO) and purified by desalting.…”

“…In our reference gene study for ovine neutrophils, YWHAZ and RPL19, along with G6PD and GAPDH, had the lowest delta Cq stability values [13]. Similar to geNorm, Norm-Finder, and BestKeeper analyses, ACTB would be excluded as a reference gene from consideration as it ranked consistently at or near the bottom four of the rank order.…”

“…In another human study, guanine nucleotide binding protein, ß-peptide 2-like1 (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]. In sheep neutrophils, we found that SDHA and glucose-6-phosphate dehydrogenase (G6PD) in healthy sheep, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and tyrosine 3-monoxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) in foot rot diseased sheep were the best pairs of references genes [13]. In ovine whole blood, SDHA and YWHAZ were considered the most suitable reference genes as they were stably expressed regardless of disease status [12].…”

“…Neutrophils use two general mechanisms to kill microorganisms: extracellular killing via neutrophil extracellular traps [7][8][9], or intracellular killing via phagocytosis followed by the secretion of antimicrobial proteins, proteolytic enzymes, and reactive oxygen species when membrane-bound granules fuse with phagocytic vesicles. We and others have reported on potential reference genes in human [10,11] and ovine neutrophils [12,13]. 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].…”