Primer extension assay to detect 2′-O-methylation of RNA

RNA was extracted from 24, 48 or 72 h treated or transfected HeLa cells with TRI-REAGENT (Cincinnati, OH, USA) according to the manufacturer's suggested protocol. 2 μg RNA was prepared with 1 μl Reverse Transcriptase buffer (New England Biolabs, Ipswich, MA, USA), 1 μl of 5 μM dig labeled primer designed to base pair downstream of the methylation site of interest (Integrated DNA Technologies, Coralville, Iowa, USA) and DEPC H₂O to 8 μl. After 2 min at 95°C and 10 min at 42°C, 1 μl Reverse Transcriptase (New England Biolabs, Ipswich, MA, USA) and 1 μl dNTPs were added and samples returned to 42°C for 1 h. The amount of dNTPs used are as noted, or were low concentrations (2.5 μM or 5 μM) used to detect ribose methylation. Samples plus loading buffer were run on a pre-warmed 15% TBE urea gel (Invitrogen) in 1× TBE at 180 V for 80 min. Gel was then rinsed in 1× TBE for 10 min. cDNA product was transferred to membrane using iBlot DNA transfer stacks (Invitrogen) with the iBlot Gel Transfer device (Life Technologies) using program 5 for 3 min, rinsed in ultrapure H₂O and crosslinked at 120 K μJ/cm². Membrane was incubated in Roche 1× blocking buffer for 15 min with slow rotation, then 30 min with slow rotation in Roche Anti-Digoxigenin-AP Fab fragments at 1:10,000 in Roche blocking buffer and washed with slow rotation in 1× wash buffer, (Roche wash and block buffer set, Roche, Mannheim, Germany). Membrane was developed with 1× CSPD in development buffer at 1:100 for 5 min at room temp, then placed between transparencies for 15 min in a 37°C incubator. Chemiluminescent images were captured and quantified with a Bio-Rad Chemi Doc Universal Hood and Quantity One Software (Bio-Rad, Hercules, CA, USA). Digoxigenin labeled DNA oligonucleotides were obtained from Integrated DNA Technologies (Coralville, Iowa, USA) and are as follows: 18S rRNA A484 site; 5′-DiGN/GCGCGCCTGCTGCCTTCCTTGGA-3′, 28S rRNA G3923 site; 5′-DiGN/CGCCGGGGGCCTCCCACTTATT-3′, 28S rRNA A2388 site; 5′-DiGN/CCCATGTTCAACTGCTGTTCAC-3′.