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.
Highlights
For many years milk somatic cell count (SCC) has been used as a major predictor of milk quality, due to its association with the inflammatory status of the mammary gland
Numerous independent experiments revealed that milk somatic cells (MSC) may be considered as a source of valuable material for gene expression studies, which may reflects the transcriptional potential of the lactating mammary gland [1,2,3]
Taking into account the fact that, to our knowledge, the present investigation is the first one, to asses relative gene expression levels in equine milk somatic cells, we decided to perform a detailed selection of potential reference genes using two bioinformatic algorithms: geNorm and NormFinder
Summary
For many years milk somatic cell count (SCC) has been used as a major predictor of milk quality, due to its association with the inflammatory status of the mammary gland. Numerous independent experiments revealed that milk somatic cells (MSC) may be considered as a source of valuable material for gene expression studies, which may reflects the transcriptional potential of the lactating mammary gland [1,2,3] It is important for the investigations focused on searching for molecular markers associated with milk composition, as well as studies regarding molecular aspects of immunological response during mammary gland inflammation [4,5]. As it was indicated by Cánovas et al (2014) [2] in comparison to the other sources (e.g. mammary gland tissue, laser dissected mammary epithelial cells), the quality of the total RNA extracted from the milk somatic cells was high. There have been a few reports describing the nucleotide sequences and the interbreed distribution of genetic polymorphisms of selected equine milk protein genes, e.g. Selvaggi et al (2010) [7], but there are no available studies focused on their relationship between the milk composition traits and the expression levels of particular genes
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