Quantification of Small RNA Transcripts

Isolated RNAs were denatured at 95°C for 3 min and separated using 15% Urea-PAGE. The gels were then stained with SYBR GOLD, imaged and immediately transferred to Roche Nylon Membranes (Roche, catalog number: 11417240001), and UV-cross-linked twice at 0.12J energy. Membranes were pre-hybridized with Roche DIG hybridization buffer (Roche, catalog number: 11796895001) for 60 min at 42°C shaker. DIG- labeled probes (5′-tRNA^Ala: 5′-DIG-CGCTCTACCACTGAGCTACACCCCC; 5′-tRNA^Val: 5′-DIG-GTGATAACCACTACACTACGGAAAC; 5′-tRNA^Gly: 5′-DIG-AATTCTACCACTGAACCACCCATGC) were added to the hybridization solution and incubated overnight at 42°C. Discarding the hybridization solution, the membranes were washed twice at 42°C with low stringent buffer (2 × SSC with 0.1% [wt/vol] SDS) for 15 min each, followed by twice washes with high stringent buffer (0.1 × SSC with 0.1% [wt/vol] SDS) for 10 min each, and finally washed by washing buffer (1 × SSC) for 10 min. Then, the membrane was blocked in a blocking buffer (Roche, catalog number: 11096176001) at room temperature for 3 h. After which, DIG antibody (Roche, Anti-Digoxigenin-AP Fab fragments, 11093274910) was added into the blocking buffer at a ratio of 1:10,000 and incubated again for another 1 h at room temperature. The membranes were washed 4 times in DIG wash buffer (1 × Maleic acid buffer, 0.3% Tween-20) at room temperature for 15 min each, followed by incubation in developing buffer for 10 min at room temperature. Finally, the membranes were coated with CSPD reagent (Roche, catalog number: 11755633001) at 37°C for 15 min in the dark and imaged by using a Bio-Rad system (USA). Original uncropped images of northern blot and denatured urea gel are shown in Additional file 9: Fig. S9.