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PloS one2015; 10(10); e0139688; doi: 10.1371/journal.pone.0139688

Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

Abstract: Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse) we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8) is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.
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Publication Date: 2015-10-05 PubMed ID: 26437076PubMed Central: PMC4593561DOI: 10.1371/journal.pone.0139688Google Scholar: Lookup
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research paper discusses the use of somatic cells from equine (horse) milk for gene expression studies, specifically the identification of the best reference genes for RT-qPCR processes. The study was carried out on milk samples from three different Polish horse breeds.

Objective

The objective of the research was to investigate whether somatic cells derived from horse milk are suitable for gene expression studies. The authors also wanted to identify genes that could serve as reliable reference points for RT-qPCR (Real-Time Quantitative Polymerase Chain Reaction), a common technique used to measure gene expression.

Methods

  • The researchers collected 39 milk samples from three different Polish horse breeds: Polish Primitive Horse, Polish Cold-blooded Horse, and Polish Warmblood Horse.
  • The focus was on extracting RNA from the somatic cells in these milk samples.
  • Seven potential reference genes for RT-qPCR were examined using two different algorithms, geNorm and NormFinder. These genes were selected due to their presumed stability.
  • The real-world application of the selected genes was validated through measuring the relative transcript abundance of the beta-casein gene, which plays a key role in milk production.

Findings

  • Even though the somatic cell count in horse’s milk was relatively low, the extracted RNA was of sufficient quantity and quality for gene expression studies.
  • There were differences in the results provided by geNorm and NormFinder algorithms when identifying the most suitable reference genes. However, KRT8 and TOP2B genes were consistently indicated as the most stable ones by both algorithms.
  • GeNorm analysis suggested a combination of four reference genes (ACTB, GAPDH, TOP2B and KRT8) was required for appropriate RT-qPCR normalization, while NormFinder identified two genes (KRT8 and RPS9) as optimal.
  • The researchers confirmed the importance of selecting the correct combination of reference genes for successful RT-qPCR experiments. Using inappropriate reference genes can distort the final results of gene expression experiments.

Conclusion

This study underscored the potential of horse milk somatic cells as a source for gene expression studies. Further, it identified a set of reference genes that can be used for more accurate RT-qPCR experiments, aiding in future research and potentially lowering the methodological errors in such studies.

Cite This Article

APA
Cieslak J, Mackowski M, Czyzak-Runowska G, Wojtowski J, Puppel K, Kuczynska B, Pawlak P. (2015). Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells. PLoS One, 10(10), e0139688. https://doi.org/10.1371/journal.pone.0139688

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 10
Issue: 10
Pages: e0139688

Researcher Affiliations

Cieslak, Jakub
  • Department of Horse Breeding, Poznan University of Life Sciences, Poznan, Poland.
Mackowski, Mariusz
  • Department of Horse Breeding, Poznan University of Life Sciences, Poznan, Poland.
Czyzak-Runowska, Grazyna
  • Department of Small Mammals Breeding and Raw Materials of Animal Origin, Poznan University of Life Sciences, Poznan, Poland.
Wojtowski, Jacek
  • Department of Small Mammals Breeding and Raw Materials of Animal Origin, Poznan University of Life Sciences, Poznan, Poland.
Puppel, Kamila
  • Department of Animal Science, Cattle Breeding Division, Warsaw University of Life Sciences, Warsaw, Poland.
Kuczynska, Beata
  • Department of Animal Science, Cattle Breeding Division, Warsaw University of Life Sciences, Warsaw, Poland.
Pawlak, Piotr
  • Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Poznan, Poland.

MeSH Terms

  • Algorithms
  • Animals
  • Female
  • Gene Expression
  • Genes, Essential
  • Horses / genetics
  • Milk / cytology

Conflict of Interest Statement

Competing Interests: The authors have declared that no competing interests exist.

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Citations

This article has been cited 9 times.
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