Article Title: OTTER, a new method quantifying absolute amounts of tRNAs
Figure Lengend Snippet: tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with dATP and fluorescence-labeled dUTP (such as TMR-dUTP; marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).
Article Snippet: After adding final concentration of 10 μM TMR-dUTP (Roche Diagnostics, Basel, Switzerland), 250 μM dATP, 10 mM MgCl2 , and 2.0 units of Klenow Fragment (3’-5’ exo− )(New England Biolabs, Ipswich, Massachusetts, USA), total 10 μl of the mixture was incubate at 37°C for 90 min.
Techniques: Fluorescence, Labeling, Polyacrylamide Gel Electrophoresis, Activity Assay, Northern Blot