Technical considerations and review of urinary microRNAs as biomarkers for chronic kidney disease in dogs and cats

MicroRNAs (miRNAs or miRs) are small, non-coding RNAs that play a crucial role in gene regulation, making them potential biomarkers for various diseases. In the field of veterinary medicine, there is a growing interest in exploring the diagnostic and therapeutic potential of miRNAs in kidney diseases affecting dogs and cats. This review focuses on the use of urinary miRNAs as biomarkers for chronic kidney disease (CKD) in these companion animals. We introduce miRNAs, their biogenesis, and their presence in biofluids, particularly within exosomes, and discuss studies investigating miRNAs in kidney tissue and urine. We acknowledge the challenges associated with miRNA studies, including preanalytical factors such as biological variation, sample collection/processing, storage conditions, and experimental design. We highlight the importance of technical considerations, such as sample pooling, sequencing depth, multiplexing, and the various steps of the miRNA experimental workflow. Furthermore, we discuss RNA isolation methods, small RNA sequencing data analysis, and the use of quantitative reverse transcription PCR (qRT-PCR) and droplet digital PCR for verification. We emphasize the importance of internal controls, spike-ins, and normalization methods to minimize technical variation and ensure reliable results in qRT-PCR analysis. This review concludes that while urinary miRNAs hold promise as non-invasive biomarkers for CKD in dogs and cats, addressing the challenges and standardization of protocols is vital for the successful translation of this research into clinical practice.