Identification of Suitable Internal Control miRNAs in Bovine Milk Small Extracellular Vesicles for Normalization in Quantitative Real-Time Polymerase Chain Reaction

Abstract

This study aimed to identify a suitable RNA extraction kit and stable internal control microRNA (miRNA) in bovine milk small extracellular vesicles (sEVs) for a quantitative polymerase chain reaction (qPCR) analysis. Two RNA extraction kits, miRNeasy Micro Kit, and Maxwell RSC miRNA Tissue Kit, were compared and evaluated using bovine milk sEVs via qPCR analysis. Five miRNAs, bta-miR-29a, bta-miR-200a, bta-miR-26b, hsa-miR-27b-3p, and hsa-miR-30b-5p, were selected by microarray analyses, and their cycle threshold (Ct) values were further evaluated mathematically using geNorm, NormFinder, BestKeeper, and ∆Ct algorithms. The results revealed that both the miRNeasy Micro Kit and Maxwell RSC miRNA Tissue Kit are useful for the efficient recovery of RNA from bovine milk sEVs. According to the final stability ranking analyzed by RefFinder, hsa-miR-27b-3p and bta-miR-29a can be used as suitable internal control miRNAs in bovine milk sEVs. The study also indicated that using a suitable internal control miRNA may improve the reliability and accuracy of the qPCR analysis for normalization in bovine milk sEVs. To the best of our knowledge, this is the first study to uncover the suitable internal control miRNAs in bovine milk sEVs.

Exploration of genes associated with induction of the viable but non-culturable state of Campylobacter jejuni

“…The selection of an internal control gene for qPCR was conducted in accordance with previous reports (Rahman et al 2021(Rahman et al , 2023 with some modifications. A total of six genes from the raw microarray data were selected for the candidate internal control gene assessment.…”

Characterization of mRNA Signature in Milk Small Extracellular Vesicles from Cattle Infected with Bovine Leukemia Virus

“…Milk sEVs were isolated and purified as described previously with slight modifications [23,24,[29][30][31]. Casein was removed from the milk using an acetic acid treatment followed by filtration of the supernatant (whey) by using 1.0-, 0.45-, and 0.2-µm pore-size filters (GA-100, C045A047A, and C020A047A, Advantec, Tokyo, Japan).…”

“…According to the MISEV2018 guidelines [17], milk sEVs were characterized by using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting (WB) as described previously [23,24,[29][30][31], with slight modifications. For TEM observation, the milk sEV pellet solution was diluted ten times using distilled water and taken onto glow-discharged carbon support films on copper grids.…”