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. 2017 Feb;23(2):161–168. doi: 10.1261/rna.058024.116

FIGURE 3.

FIGURE 3.

TaqMan qRT-PCR method to analyze the 5′-terminal nucleotide of mature tRNAHisGUG. (A) Schematic representation of the TaqMan qRT-PCR analysis used to quantify each 5′-terminal variant of mature tRNAHisGUG. (B) Sequences and/or positions of the mature tRNAHisGUG and the following TaqMan qPCR components: adapter, AS-disrupter, primers, and TaqMan probe. (C) Under the indicated conditions, this method was applied to synthetic mature tRNAHisGUG containing G−1. The reaction containing only T4 RNA ligase (far left) was set to one, and fold changes relative to this reference are shown; bars indicate SD from three independent experiments. Amplified cDNA bands observed in native PAGE after 40 cycles of PCR are also shown. (D) Synthetic mature tRNAsHisGUG starting from G1, G−1, and U−1 were mixed at the indicated ratios and quantified by the TaqMan qRT-PCR. Detected amounts were calculated using standard curves (Supplemental Fig. S5), and the relative abundances of detected tRNAs containing each 5′-terminal nucleotide are shown. Bars indicate SD from three independent experiments. N.D. indicates that the reaction did not amplify detectable cDNA signals.