Rahman Md Matiur, Takashima Shigeo, Kamatari Yuji O, Badr Yassien, Shimizu Kaori, Okada Ayaka, Inoshima Yasuo
The United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Laboratory of Food and Environmental Hygiene, Cooperative Department of Veterinary Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Membranes (Basel). 2021 Nov 26;11(12):933. doi: 10.3390/membranes11120933.
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".
牛乳小细胞外囊泡（sEVs）含有许多具有重要生物学意义的分子，包括mRNA。定量实时聚合酶链反应（qRT-PCR）是一种广泛用于定量组织和细胞中mRNA的方法。然而，尚未确定用于牛乳sEVs中qRT-PCR标准化的合适假定内参基因的使用、选择和稳定性。因此，本研究的目的是确定牛乳sEVs中用于qRT-PCR数据标准化的合适假定内参基因。从六头健康的荷斯坦-弗里生奶牛中分离出牛乳sEVs，随后进行RNA提取和cDNA合成。总共选择了17种mRNA，使用qRT-PCR进行研究和定量；使用geNorm、NormFinder、BestKeeper和∆CT算法对它们进行进一步评估，以确定那些在牛乳sEVs中高度稳定的假定内参基因。使用RefFinder确定合适假定内参基因的最终排名。通过qRT-PCR在六头牛的牛乳sEVs中检测到来自TUB、ACTB、DGKZ、ETFDH、YWHAZ、STATH、DCAF11和EGFLAM的mRNA。RefFinder表明，TUB、ETFDH和ACTB在牛乳sEVs中高度稳定，因此适用于qRT-PCR数据的标准化。本研究表明，使用这些基因作为假定内参基因可能会进一步提高qRT-PCR在牛乳sEVs中的稳健性。由于这些假定内参基因适用于健康的牛，在“正常或健康条件下”说明这些基因在sEVs中是稳定的将是有帮助的。
