# Stability of microRNAs in Canine Serum—A Prerequisite for Use as Biomarkers in Tumour Diagnostics

## TL;DR

This study examines how stable microRNAs are in dog blood under different storage conditions to determine their potential as cancer biomarkers.

## Contribution

The study provides new insights into the stability of specific miRNAs in canine serum under various storage conditions.

## Key findings

- miR-21 was the most abundant, while miR-494 was the least in canine blood samples.
- miR-192 showed the best stability, while miR-21, miR-122, and miR-222 significantly declined after 48 hours.
- Standardisation of miRNA collection and analysis is essential for reliable canine cancer detection.

## Abstract

Since the levels of miRNAs differ between healthy and neoplastic patients and can be detected in the bloodstream, they are investigated as potential bio markers for cancer. To be useful in routine diagnostics, miRNAs must remain stable in blood samples over time. For stability evaluation, serum samples from 10 healthy dogs were analysed, measuring the levels of eight different miRNAs under various storage conditions (4 °C and 20 °C for 24 and 48 h). Results showed that miR-21 was the most abundant, while miR-494 was the least. The miRNA levels varied among individual dogs, sometimes by 5- to 10-fold. Some miRNAs, like miR-192, remained stable, while others, such as miR-21, miR-122, and miR-222, significantly declined after 48 h. These findings highlight the importance of considering the stability of the various miRNAs when using them as diagnostic biomarkers. Standardising miRNAs’ collection and analysis methods is essential to ensure reliable results in canine cancer detection by liquid biopsy.

Since microRNAs are released into the bloodstream and miRNA profiles are supposed to differ between healthy and tumour patients, miRNAs seem to have potential as biomarkers. An essential prerequisite for biomarkers in a routine diagnostic setup is their stability in serum over time. In this study, serum samples from 10 healthy dogs were analysed at different time points and under various temperature conditions (after 24 and 48 h, at 4° or 20 °C) for the copy number of eight miRNAs (miR-20b, 21, 122, 126, 192, 214, 222, 494) using ddPCR. The miR-21 had the highest copy number, whereas miR-494 had the lowest copy number in canine blood samples. The values of each miRNA varied individually between the dogs, showing a 5 to 10-fold range. Stability differed between the miRNAs, with miR-192 having the best stability. The copy number of miR-20b, miR-126 and miR-214 decreased not significantly during 48 h storage time. In contrast, miR-21, miR-122 and miR-222 were stable for 24 h only but decreased significantly after 48 h. The (in)stability of individual canine miRNAs must be considered when transferring study results into veterinary routine diagnostics, as the transport and storage conditions are variable. As far as possible, standardisation of sampling, storage and quantification of miRNAs is needed.