Quantitative RNA Nucleoside Analysis

Three-microgram to 100-ng portions of RNA were digested to single nucleosides with AP (0.2 U; Sigma-Aldrich, St. Louis, MO, USA), phosphodiesterase I (0.02 U; VWR, Radnor, PA, USA), and Benzonase (0.2 U) in tris (pH 8, 5 mM)– and MgCl₂ (1 mM)–containing buffer. Furthermore, tetrahydrouridine (0.5 μg from Merck), butylated hydroxytoluene (1 μM), and pentostatin (0.1 μg) were added to protect modification (45 (link)). The mixture was incubated with the RNA for 2 hours at 37°C. Afterward, samples were filtered through 96-well filter plates (AcroPrep Advance 350 10K Omega, Pall Corporation, NY, USA) for longer than 10 min at 3000g to remove digestive enzymes.