title: 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.
authors.affiliation: Department of Horse Breeding, Poznan University of Life Sciences, Poznan, Poland.
authors.forename: Jakub
authors.initials: J
authors.lastname: Cieslak
authors.affiliation: Department of Horse Breeding, Poznan University of Life Sciences, Poznan, Poland.
authors.forename: Mariusz
authors.initials: M
authors.lastname: Mackowski
authors.affiliation: Department of Small Mammals Breeding and Raw Materials of Animal Origin, Poznan University of Life Sciences, Poznan, Poland.
authors.forename: Grazyna
authors.initials: G
authors.lastname: Czyzak-Runowska
authors.affiliation: Department of Small Mammals Breeding and Raw Materials of Animal Origin, Poznan University of Life Sciences, Poznan, Poland.
authors.forename: Jacek
authors.initials: J
authors.lastname: Wojtowski
authors.affiliation: Department of Animal Science, Cattle Breeding Division, Warsaw University of Life Sciences, Warsaw, Poland.
authors.forename: Kamila
authors.initials: K
authors.lastname: Puppel
authors.affiliation: Department of Animal Science, Cattle Breeding Division, Warsaw University of Life Sciences, Warsaw, Poland.
authors.forename: Beata
authors.initials: B
authors.lastname: Kuczynska
authors.affiliation: Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Poznan, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Pawlak
mesh_terms: D000465:Algorithms; D000818:Animals; D005260:Female; D015870:Gene Expression; D020043:Genes, Essential; D006736:Horses / Q000235:genetics*; D008892:Milk / Q000166:cytology*