Vacuette z serum sep clot activator

From May 2020 to January 2022, a total number of 174 serum samples have been processed in a prospective study. The research protocol has been developed in a clinical laboratory certified according to ISO 9001:2015.
Samples are drawn in gel separator tubes without anticoagulant (Vacuette Z serum sep clot activator, Greiner Bio-One, Kremsmünster, Austria). Samples used in this study are obtained from out-patients referred to the laboratory for blood testing and from in-patients. After laboratory testing is done, remaining serum samples are used for the study purposes. No additional blood sampling was needed for this research.
The following biochemical analytes have been evaluated in this project: albumin, alkaline phosphatase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), calcium, creatinine, gamma-glutamyltransferase (GGT), glucose, phosphates, total proteins, urea and total bilirubin. Since not all the considered analytes were measured in the whole set of 174 serum samples, a specific number of measurements was obtained for each specific analyte.
The study has been approved by the Hospital’s ethics committee of clinical research (CEIM code:2020672).

Blood sampling was taken following the routine health check protocol in the Gdf training program. During the GdF veterinarian procedures, the handler asked the dog to stand and stay still for 1 min while gently manipulating and distracting it. Next, the handler asked the dog to sit; simultaneously, the veterinarian collected the 3 mL of blood sample via the radial vein into Vacuette Z Serum Sep Clot Activator (GREINER BIO-ONE). After centrifugation (2000 × g, 10 min), serum was obtained and stored at − 80 °C until use. Hemolysis was controlled in all serum samples to prevent the release of microRNA contained in the blood cells altered ecmiRNA profile (SM 2. Hemolysis assessment during sample preparations). Each serum sample was separated into two collection tubes: one tube (300 µL) for analyzing biochemical parameters and one (200 µL) to explore differential miRNA expression profiles. Total RNA, including ecmiRNAs, was extracted from 200 µL of serum using the miRNeasy Serum/Plasma Kit (QIAGEN CLC bio, Aarhus, Denmark) according to the manufacturer’s instructions, with an elution volume of 14 µL (SM 3. RNA extraction and Spike-in for qPCR validations) and then stored at − 80 °C until use. MiRNA concentration was assessed using the Qubit Fluorometer 4 and the Qubit microRNA Assay Kit (THERMOFISHER SCIENTIFIC, Kandel, Germany).