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

Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils

1email, 1,†email, 2,3email and 1,* email
Received: 30 March 2013; in revised form: 15 May 2013 / Accepted: 15 May 2013 / Published: 30 May 2013
(This article belongs to the Section Molecular Diagnostics)
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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
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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.

AMA Style

Vorachek WR, Hugejiletu, Bobe G, Hall JA. Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils. International Journal of Molecular Sciences. 2013; 14(6):11484-11495.

Chicago/Turabian Style

Vorachek, William R.; Hugejiletu; Bobe, Gerd; Hall, Jean A. 2013. "Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils." Int. J. Mol. Sci. 14, no. 6: 11484-11495.


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