Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.
Authors: Cieslak Jakub, Mackowski Mariusz, Czyzak-Runowska Grazyna, Wojtowski Jacek, Puppel Kamila, Kuczynska Beata, Pawlak Piotr
Journal: PloS one
Summary
# Editorial Summary Milk somatic cells offer a non-invasive source of mRNA for investigating genetic expression in mares, yet establishing appropriate normalisation methods has been lacking in equine research. Researchers analysed 39 milk samples from three Polish horse breeds, extracting RNA from somatic cells and testing seven candidate reference genes using two stability algorithms (geNorm and NormFinder) to identify the most reliable internal controls for reverse-transcription quantitative PCR. Despite mares' characteristically low somatic cell counts, RNA quantity and quality proved adequate for gene expression work; notably, KRT8 and TOP2B demonstrated superior stability across both analytical approaches, though the two algorithms diverged on optimal reference gene combinations—geNorm recommending four genes (ACTB, GAPDH, TOP2B and KRT8) whilst NormFinder favoured just two (KRT8 and RPS9). For equine professionals involved in breeding decisions, performance optimisation or mastitis management, this work establishes a validated framework for conducting meaningful gene expression studies on milk samples, emphasising that careless selection of normalisation genes can substantially skew real-time PCR results and potentially lead to incorrect biological conclusions.
Read the full abstract on PubMed
Practical Takeaways
- •Equine milk somatic cells are a viable source for gene expression research despite their lower cell counts compared to other species
- •If conducting RT-qPCR studies on equine milk, use either the 4-gene combination or KRT8+RPS9 pairing as reference genes depending on your analytical platform
- •Always validate your chosen reference genes using multiple algorithms before proceeding with full expression studies to ensure reliable and reproducible results
Key Findings
- •Despite low somatic cell counts in mare's milk, RNA quantity and quality are sufficient for gene expression studies
- •KRT8 and TOP2B genes demonstrated the highest stability across both geNorm and NormFinder algorithms
- •geNorm recommended a 4-gene reference panel (ACTB, GAPDH, TOP2B, KRT8) while NormFinder identified KRT8 and RPS9 as optimal combination
- •Proper reference gene selection significantly influences RT-qPCR results and must be carefully validated for each experiment