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Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells
Jakub Cieslak1, Mariusz Mackowski1, Grazyna Czyzak-Runowska2
1Department of Horse Breeding, Poznan University of Life Sciences, Poznan, Poland.
Plos One
|October 6, 2015
Summary
Equine milk somatic cells are a viable source for gene expression studies, even with low cell counts. KRT8 and TOP2B emerged as the most stable reference genes for quantitative real-time PCR (RT-qPCR) normalization in mare
Area of Science:
- Animal Science
- Molecular Biology
- Dairy Science
Background:
- Somatic cell count in milk indicates mammary gland inflammation.
- Milk somatic cells are valuable for mammalian gene expression studies.
- Mare's milk consumption is increasing, driving interest in its genetic variability.
Purpose of the Study:
- To assess the suitability of equine milk somatic cells for mRNA extraction and gene expression analysis.
- To identify the most stable reference genes for normalizing quantitative real-time PCR (RT-qPCR) data in mare's milk somatic cells.
- To investigate the impact of reference gene selection on gene expression results.
Main Methods:
- Collected 39 milk samples from three Polish horse breeds.
- Extracted RNA from milk somatic cells and assessed its quality and quantity.
- Evaluated 7 potential reference genes using geNorm and NormFinder algorithms for RT-qPCR normalization.
- Performed trial studies using different reference gene combinations to analyze beta-casein gene expression.
Main Results:
- RNA extracted from mare's milk somatic cells was sufficient for gene expression studies, despite low cell counts.
- KRT8 and TOP2B were identified as the most stable reference genes by both geNorm and NormFinder algorithms.
- geNorm recommended a combination of ACTB, GAPDH, TOP2B, and KRT8, while NormFinder suggested KRT8 and RPS9 for optimal normalization.
- The choice of reference gene(s) significantly impacted the relative transcript abundance results.
Conclusions:
- Equine milk somatic cells are a practical source for gene expression analysis.
- Careful selection and validation of reference genes are critical for accurate RT-qPCR results in mare's milk studies.
- KRT8 and TOP2B are promising candidates for reference genes in equine milk somatic cell expression studies.
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