Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils

Public Deposited

Downloadable Content

Download PDF


Attribute NameValues
  • 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: Whole blood, Footrot, Normalization, Immune responses, Selenium, Real time PCR, Expression, Housekeeping genes
Resource Type
Date Available
Date Issued
  • Vorachek, W. R., Hugejiletu, Bobe, G., & Hall, J. A. (2013). Reference gene selection for quantitative PCR studies in sheep neutrophils. International Journal of Molecular Sciences, 14(6), 11484-11495.
Journal Title
Journal Volume
  • 14
Journal Issue/Number
  • 6
Academic Affiliation
Rights Statement
Funding Statement (additional comments about funding)
  • Funded in part by USDA CSREES 2008-35204-04624, Agricultural Research Foundation andAnimal Health and Disease Project Formula Funds, Oregon State University, Corvallis, OR, USA.
Peer Reviewed



This work has no parents.