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.

Lactose hydrolysis and lactase activity in fermented mixtures containing mare's, cow's, sheep's and goat's milk

“…In turn, raw MM contained 18.2 × 10 3 TBC mL −1 and 23.4 × 10 3 SCC mL −1 . Recorded values concerning the cytological and microbiological quality of milk collected for analyses may be considered good and consistent with literature data reported by other authors for high‐quality processed milk (Danków et al ., ; Skrzypek et al ., ; Cieślak et al ., ; Markiewicz‐Kęszycka et al ., ).…”

Variability of lysozyme and lactoferrin bioactive protein concentrations in equine milk in relation to LYZ and LTF gene polymorphisms and expression

“…Obtained results were normalized to the geometric mean of four internal reference genes: ACTB , GAPDH , TOP2B (housekeeping genes) and KRT8 (marker of the mammary epithelial cell number). Selection of the above‐mentioned reference genes was made on the basis of the geNorm algorithm as described in detail in our previous article …”

“…Selection of the above-mentioned reference genes was made on the basis of the geNorm algorithm as described in detail in our previous article. 16…”

Genes encoding equine β-lactoglobulin (LGB1 and LGB2): Polymorphism, expression, and impact on milk composition

“…Total RNA from milk somatic cells (MSC) was obtained by extraction using TriPure Isolation Reagent (Roche, USA), according to a previously described procedure [26]. cDNA synthesis was performed with a Transcriptor High Fidelity cDNA Synthesis Kit (Roche, USA) according to manufacturer's instructions.…”

“…The relative transcript level measurements using a LightCycler 480 real-time PCR instrument (Roche, USA) were carried out in duplicate for the LGB1 and LGB2 genes and four reference genes (ACTB, GAPDH, TOP2B, and KRT8). Selection of internal control genes was conducted using the GeNorm and NormFinder tools, as previously described [26]. Primers and TaqMan probe sequences for real-time PCR were designed and synthesized by TIB MolBiol (Germany) (S1 Table).…”