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. 2024 Jun;30(6):739–747. doi: 10.1261/rna.079895.123

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© 2024 Zhang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.

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FIGURE 4. — Measuring m¹A58 modification in biological RNA. (A) The sequence of yeast tRNA^Phe and oligos used in TL-qPCR for m¹A58 modification (″: bold and underlined). The two pairs of linkers for yeast tRNA^Phe-58m¹A and for -5p (control) contain sequences for Cy5 and TR (Texas Red) probe binding sites for qPCR. (B) Cq values of m¹A (red) or control (black) ligation oligos of serial diluted inputs of yeast tRNA^Phe from 3.1 to 800 fM without AlkB treatment. (C) Differential Cq values between m¹A and control ligation oligos of serial diluted yeast tRNA^Phe samples without (black) and with (red) AlkB demethylase (DM) treatment. (D) Fraction of m¹A58 in yeast tRNA^Phe as measured by TL-qPCR using varying amounts of input RNA. (E) Oligonucleotides and linkers used in SplintR ligation for the m¹A58 site of human tRNA_i^Met and tRNA^His, tRNA^Trp is used as an internal control. Three-color qPCR is run using either HEX(m¹A-iMet) + TR(m¹A-His) + FAM(Trp) or HEX(CTRL-iMet) + TR(CTRL-His) + FAM(Trp) combination. (F) ΔCq values of 5lkrT (m¹A) and 5lkrT + 4 (CTRL) on tRNA_i^Met (HEX probe), tRNA^His (TR probe), and tRNA^Trp (FAM probe) without (DM−) and with AlkB treatment (DM+). (G) m¹A58 fraction measured by TL-qPCR for tRNA_i^Met and for tRNA^His. Average of n = 3 biological replicates. Inset: modification index (MI) around the m¹A58 (±5 nt) site measured by DM-tRNA-seq for tRNA_i^Met and tRNA^His (sequencing data from NCBI GEO GSE66550).