Northern Blotting of mt tRNA Leu(UUR)

Northern blotting analysis of mt tRNA^Leu(UUR) was conducted essentially as described previously (33 (link)). Total RNA (3 μg) from cultured cells was dissolved in 10% PAGE containing 7 M urea and stained with ethidium bromide before blotting onto a nylon membrane (Amersham Hybond N+; GE Healthcare Systems). A synthetic DNA probe for mt tRNA^Leu(UUR) was 5′-labeled with [γ-³²P]ATP (PerkinElmer, Inc., Waltham, MA) by T4 Polynucleotide Kinase (Toyobo, Osaka, Japan). The following two types of probes were used: probe 1 (5′-TGTTAAGAAGAGGAATTGAACCTCTGACTG-3′) for comparing 2SA cells with 2SD cells, and probe 2 (5′-TTTTATGCGATTACCGGGCCCTGCCATCTT-3′) for comparing 2SD cells with 2KD cells. The radioactivity on the membrane was visualized by exposing the membrane to an imaging plate and analyzed using an FLA-7000 imaging analyzer (Fujifilm). The 5S rRNA was detected by ethidium bromide staining.