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. 2023 Mar 17;51(14):7563–7579. doi: 10.1093/nar/gkad139

Figure 2.

Figure 2.

Measuring τm5U frequency in mt-tRNALeu(UUR) by CMC-PE. (A) Schematic of CMC-PE. The detailed procedure is described in the ‘MATERIALS AND METHODS’ section. (B) cDNA bands of CMC-PE for τm5U34 of mt-tRNALeu(UUR) in total RNA mixtures of HeLa and GTPBP3 KO cells at different mixing ratios. CMC-PE for the MELAS patient myoblasts bearing 3243A > G (MC cell line) was also performed. When mt-tRNALeu(UUR) has no τm5U34, the cDNA stops at position 32 through the insertion of dideoxyguanosine (red arrow). When mt-tRNALeu(UUR) is fully modified with τm5U34, the cDNA is stopped at positions 33 (blue arrow) and 35 (green arrow) by CMC-τm5U34. A part of primer is indicated by the yellow line. τm5U frequency and band intensity ratio for each lane are shown below the gel image. (C) Calibration line of CMC-PE for τm5U34 of mt-tRNALeu(UUR) in total RNA mixtures of HeLa and GTPBP3 KO cells at different mixing ratios. The band intensity ratios were calculated as described in ‘MATERIALS AND METHODS.’ Data represent the average values of technical triplicates ± s.d. (R2= 0.9995).