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Int. J. Mol. Sci. 2013, 14(6), 11484-11495; doi:10.3390/ijms140611484

Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils

1
Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA
2
Department of Animal and Rangeland Sciences, College of Agricultural Sciences, Oregon State University, Corvallis, OR 97331, USA
3
Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA
Current address: College of Veterinary Medicine, Inner Mongolian Agricultural University, Hohhot 010018, China
*
Author to whom correspondence should be addressed.
Received: 30 March 2013 / Revised: 15 May 2013 / Accepted: 15 May 2013 / Published: 30 May 2013
(This article belongs to the Section Molecular Diagnostics)
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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.
Keywords: blood neutrophils; foot rot; Ovis aries; qPCR; reference genes; selenium treatment blood neutrophils; foot rot; Ovis aries; qPCR; reference genes; selenium treatment
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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MDPI and ACS Style

Vorachek, W.R.; Hugejiletu; Bobe, G.; Hall, J.A. Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils. Int. J. Mol. Sci. 2013, 14, 11484-11495.

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