Putative Internal Control Genes in Bovine Milk Small Extracellular Vesicles Suitable for Normalization in Quantitative Real Time-Polymerase Chain Reaction

Abstract

Bovine milk small extracellular vesicles (sEVs) contain many biologically important molecules, including mRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for quantifying mRNA in tissues and cells. However, the use, selection, and stability of suitable putative internal control genes in bovine milk sEVs for normalization in qRT-PCR have not yet been identified. Thus, the aim of the present study was to determine suitable putative internal control genes in milk sEVs for the normalization of qRT-PCR data. Milk sEVs were isolated from six healthy Holstein-Friesian cattle, followed by RNA extraction and cDNA synthesis. In total, 17 mRNAs were selected for investigation and quantification using qRT-PCR; they were further evaluated using geNorm, NormFinder, BestKeeper, and ∆CT algorithms to identify those that were highly stable putative internal control genes in milk sEVs. The final ranking of suitable putative internal control genes was determined using RefFinder. The mRNAs from TUB, ACTB, DGKZ, ETFDH, YWHAZ, STATH, DCAF11, and EGFLAM were detected in milk sEVs from six cattle by qRT-PCR. RefFinder demonstrated that TUB, ETFDH, and ACTB were highly stable in milk sEVs, and thus suitable for normalization of qRT-PCR data. The present study suggests that the use of these genes as putative internal control genes may further enhance the robustness of qRT-PCR in bovine milk sEVs. Since these putative internal control genes apply to healthy bovines, it would be helpful to include that the genes were stable in sEVs under “normal or healthy conditions”.

Identification of Potential mRNA Biomarkers in Milk Small Extracellular Vesicles of Enzootic Bovine Leukosis Cattle

“…Nanoparticle size analysis showed that the peak of nanoparticle size distribution was approximately 100 nm in all milk sEVs ( Figure 1 B). These results confirm the presence of milk sEVs in this study [ 28 , 29 , 34 , 35 ].…”

“…Isolation and purification of milk sEVs was carried out as previously described [ 23 , 28 , 29 ], with slight modifications. Briefly, after removing the milk fat by centrifugation at 2000× g for 20 min using an A2506 centrifuge (Kubota, Tokyo, Japan), defatted milk was preheated at 37 °C for 10 min.…”

“…The sEV suspension was diluted to 1:100 with 0.1 µm-filtrated water, followed by filtration with a 0.22 µm filter and the nanoparticle size distribution was analyzed using a Zetasizer Nano ZS nanoparticle analyzer (Malvern Panalytical, Worcestershire, UK). Isolated milk sEVs were confirmed by detecting sEV surface and internal marker proteins MFGE8 and HSP70 by Western blot analysis as described previously [ 28 , 29 ]. Anti-MFGE8 monoclonal antibody (1:10,000, clone 6F11, a kind gift from Dr. Tsukasa Matsuda, Fukushima University, Japan) and anti-HSP70 monoclonal antibody (1:100, ADI-SPA-820, Enzo Life Science, Farmingdale, NY, USA) were used as primary antibodies, and anti-mouse IgG, HRP-linked antibody (1:2,000, #7076, Cell Signaling Technology, Danvers, MA, USA) was used as a secondary antibody.…”

“…RNA extraction from milk sEVs was performed as described previously [ 29 ], with slight modifications. mRNA in sEVs was extracted using Maxwell RSC (AS4500, Promega).…”

“…The program was as follows: 50 °C for 2 min for PCR initial heat activation, followed by 40 cycles of 95 °C for 3 s for denaturation, and 60 °C for 30 s for annealing and extension. Amplification of ACTB mRNA was performed for each sample to normalize the encapsulation of the selected mRNAs [ 29 ]. After amplification, melt curve analysis was performed to validate the specificity of the reactions.…”