Bovine Blood Sampling and RNA Extraction

Peripheral blood samples (3 ml) were collected from the tail vein of cattle with the BD Vacutainer® Plus Plastic K₂EDTA Tubes and BD Vacutainer® Plus Plastic Serum Tubes. The extraction of total RNA from whole blood was performed as previously described [26 (link)]. In brief, 125 μl of whole blood was mixed with the same volume of RNase-free water and 750 μl of Trizol LS reagent (Ambion) and incubated at room temperature for 5 min. Thereafter, 200 μl of chloroform (Sigma-Aldrich) was mixed and centrifuged at 13,523 ×g and 4°C for 15 min. The supernatant was collected into a 1.5 ml tube, mixed with the same volume of 70% ethanol, and then transferred to an RNAeasy column (Qiagen, Hilden, Germany) and centrifuged at 8,500 ×g for 15 sec. After the wash steps, 30 μl of RNase-free water was added and centrifuged at 8,500 ×g for 1 min. Eluted RNA was stored at -80°C until use. For the separation of serum, 3 ml of blood samples were centrifuged at 1,500 ×g for 10 min. Separated serum was transferred to 1.5 ml tube and analyzed for the presence of MAP-specific antibodies using a commercial ELISA kit.