Bioinformatic Analysis of Bovine Uterine Extracellular Vesicles miRNA for Detection of Stable Internal Control

The present study aimed to identify stable internal control miRNAs in the bovine uterine EVs studies, resolving a critical gap in extracellular vesicle research. During follicular, luteal, and pregnancy stages, uterine fluids were collected from cows (n = 5 per stage), and EVs were isolated via sequential centrifugation and a commercial kit. Small RNA sequencing detected 464, 429, and 434 miRNAs in follicular, luteal, and pregnancy stages, respectively. Candidate miRNAs within 25% of the mean TPM value were selected (miR-200c, let-7d, miR-125b, miR-93, miR-223, and miR-224), and U6 was added to them, then analyzed by qPCR. Stability was evaluated using NormFinder, geNorm, BestKeeper, and ΔCt algorithms. After ranking in each analysis, the Geometric Mean was calculated to get the final rank. The miRNA with the best stability ranking was miR-200c (1.41), followed by let-7d (2.12), and the lowest was U6 (7.00). This is the first study to validate miR-200c and let-7d as reliable internal controls for miRNA quantification in bovine uterine EVs, addressing the absence of standardized references. These findings enhance accuracy in future research on EVs miRNAs regulating bovine reproductive functions.