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Published on: January 19, 2017
Reference gene selection for quantitative PCR studies in sheep neutrophils
William R Vorachek1, Hugejiletu, Gerd Bobe
1Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA. Jean.Hall@oregonstate.edu.
International Journal of Molecular Sciences
|June 1, 2013
Summary
Selecting appropriate reference genes is crucial for accurate mRNA expression analysis in sheep neutrophils. This study identified G6PD, YWHAZ, GAPDH, RPL19, and SDHA as the most stable genes under foot rot and Se supplementation conditions.
Area of Science:
- Molecular Biology
- Animal Science
- Biochemistry
Background:
- Quantitative PCR (qPCR) relies on stable reference genes for accurate mRNA expression analysis.
- Neutrophils are critical immune cells, and their gene expression requires validated reference genes, especially under disease conditions.
Purpose of the Study:
- To identify and validate stable reference genes for quantitative PCR (qPCR) analysis in sheep neutrophils.
- To evaluate reference gene stability under experimental conditions of foot rot (FR) and selenium (Se) supplementation.
Main Methods:
- Investigated 11 candidate reference genes in sheep neutrophils.
- Screened genes based on expression level and variability.
- Assessed expression stability using geNorm, NormFinder, BestKeeper, and delta Cq methods.
Main Results:
- G6PD, YWHAZ, GAPDH, RPL19, and SDHA were identified as the most stable reference genes.
- SDHA stability was compromised in FR-diseased sheep with Se treatment.
- Commonly used genes like PGK1, ACTB, and B2M proved unreliable.
Conclusions:
- Reference gene validation is essential for specific experimental conditions in sheep neutrophils.
- Using multiple reference genes enhances the robustness of qPCR results.
- Optimal reference gene pairs were identified for healthy (SDHA/G6PD) and diseased (GAPDH/YWHAZ) sheep.
