SAR trial. The simulated SAR trial required dogs to find a target odor (a hidden operator and his breath, simulating a missing person) on a rubble field (30 × 35 m), within a maximum time of 15 min. Blood was collected at T0 immediately before the SAR (resting baseline) and at T1 later in the SAR (physiological stress). T0 versus T1 enables a relation between two different physiological states, where T1 comprises a multifactorial combination of stresses physiologically recovered in trained dogs [15 (link)].
The experimental framework of the study is divided into two phases.

In the first step, dog blood was drawn at T0 (n = 22) and T1 (n = 22) (dogs reported in Table 1), and total serum RNA was isolated. The total RNA of six dogs listed in Table 1 (dog ID: A, B, C, D, E, and F) was used for library preparation and NGS miRNome sequencing. This step aimed to evaluate the T0 versus T1 unregulated circulating miRNAs, i.e., candidate stable EC miRNAs.

In the second step, unregulated cmiRNAs selected as candidate EC were validated by qPCR in a larger panel of dogs, including all dogs listed in Table 1 (T0 n = 22; T1 n = 22). Then, bioinformatic analysis of the qPCR results identified the EC miRNA stability values and the EC final ranking (Figure 1).

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