OvHV2 miRNA Identification
OvHV2 miRNA Identification
BJ1035 cells were grown in suspension in T175 flasks (Nunc) in 60ml of Iscoves Modified Dulbeccos Medium (IMDM) (Invitrogen) supplemented with 10% foetal calf serum (Sera Laboratories International) 1% penicillin-streptomycin (Invitrogen) 0.05% interleukin 2 (IL2) (Novartis Pharmaceuticals UK Ltd) and incubated at 37C, 5% CO2.  Once these suspension cells had reached a high concentration, the cells were pipetted up and down to break up clumps and 2/3 of the suspension was transferred to a new T175 flask with 1/3 fresh medium. 5x106 BJ1035 cells were isolated and centrifuged at 478xg for 10 minutes at room temperature to pellet the cells.  The supernatant was removed, the pellet resuspended in the remaining media, and then transferred to a 1.5ml microcentrifuge tube on ice (Greiner Bio-One).  The cells were centrifuged at 9211xg for 30 seconds at room temperature (DJB, Sigma 1-13) and the remaining media was removed.  1ml of RNA-Bee (AMS Biotechnology [Europe] Ltd) was used to homogenize the cells and degrade endogenous RNases by pipetting up and down.  200?l chloroform (Sigma) was added to the lysate, which was then shaken vigorously by hand for 15-30 seconds, stored on ice for 5 minutes, and centrifuged at 12000xg for 15 minutes at 4C (DJB, Sigma 1-15PK).  The RNA-containing aqueous phase was removed to a clean 1.5ml microcentrifuge tube and was treated with 500?l isopropanol (Sigma) to desalt and bring the RNA out of solution.  The solution was allowed to sit at room temperature for 5-10 minutes and then centrifuged at 12000xg for 5 minutes at 4C to pellet the RNA.  The supernatant was removed and the RNA pellet was washed with 200?l 75% ethanol, made with 0.1% diethylpyrocarbonate (DEPC)-treated water (1ml DEPC in 1L water, left overnight at room temperature and then autoclaved to deactivate the DEPC; Sigma) to remove any remaining salt after the isopropanol precipitation.  The sample was vortexed briefly to dislodge the pellet from the side of the tube to ensure complete washing, and was centrifuged at 7500xg for 5 minutes at 4C to re-pellet.  The ethanol was removed and the RNA pellet was allowed to air dry for 5-10 minutes on ice before being re-solubilised in 50?l 0.1% DEPC-treated water, by pipetting up and down.
Experimental Factor: ORGANISM
ovine herpesvirus 2