Optimized RNA ligation conditions achieve high efficiency for both 3′ and 5′ ligation steps. (A) 3′ ligation efficiencies were determined by resolving ligation reactions with radiolabeled synthetic miRNA substrates and Linker-3 (Integrated DNA Technologies) on 15% Urea-PAGE. A dash indicates free miRNA; a dash with a box indicates 3′ ligation product. The numbers at the bottom indicate the percentage of ligated species relative to the total. (B) 3′ ligation efficiencies were tested as in (A) with varying 3′ linkers and 10% w/v PEG8000. (C) 3′ ligation efficiency was enhanced by increasing the amount of NN linker used. The numbers at the top indicate fold molar excess of linker to substrate RNA. (D) A mixture of 29 synthetic miRNAs were radiolabeled and subjected to enhanced 3′ ligation conditions in the presence of 0.5 μg of total RNA obtained from mouse skin. The numbers at top indicate time in hours the reaction was carried out. (E) 5′ ligation efficiencies were assessed using gel-purified, radiolabeled, 3′ ligated, equimolar 29 synthetic miRNA mixtures with T4 RNL1 (left panel) at room temperature or with thermostable (TS) RNA ligase at 60°C (right panel) for 2 hours. The numbers at the bottom indicate the ligation efficiency, and a dash between two boxes indicates 5′ ligation product. (F) Optimized 5′ ligation reaction achieves 96% ligation efficiency.