Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood

Abstract

The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells.

Reference gene selection for quantitative PCR studies in bovine neutrophils

“…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]. In the bovine, potential reference genes have been evaluated in polymorphonuclear leukocytes [14], mammary epithelial cells [15], adipose tissue [16], milk somatic cells [17], and WB-cells [18] of lactating dairy cows, and in muscular tissue [19]; however, to our knowledge, potential reference genes have not been evaluated in WB-neutrophils under conditions of Se supplementation in growing beef calves.…”

“…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.…”

“…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].…”

Refining a ‘toolkit’ for objective assessment of pain and stress in ruminants

“…The relative expression of target genes IL-6 and POMC were calculated after their Ct threshold had been normalised to its reference genes within each sample. Although YWHAZ and SDHA are genes recommended for normalisation in sheep blood due to their stability in various diseased animals (Peletto et al, 2011;Vorachek et al, 2013), it has been reported that this may not be consistent in all conditions (Bustin, 2002). In the present study, the expression of SDHA and YWHAZ genes were low in some samples, which implied the lack of starting material or that RNA may have been lost during the RNA isolation and purification as well cDNA synthesis (Bustin, 2002).…”

“…Published primer pairs sequence for ovine IL-6, POMC, tyrosine 3-monoxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) and succinate dehydrogenase complex subunit A (SDHA) genes were used (Table 3.1) and were synthesised by GeneWorks (Australia). The YWHAZ and SDHA genes had been described as good reference genes that are not affected by disease or heat stress in sheep blood (Peletto et al, 2011;Vorachek et al, 2013), and therefore the genes were included in all real-time PCR analyses as housekeeping genes for data normalisation. Samples containing 2 µL cDNA, 1 µM of each forward and reverse primers, and SYBR ® Green PCR master mix, were added into a 100-well gene disc in triplicate using the Corbett CAS-1200 automated workstation (QIAGEN Pty Ltd, Chadstone, Victoria, Australia).…”