Qpcr master mix

The sequences of the adapter, AS-disrupter, primers, and TaqMan probes for TaqMan qRT-PCR are shown in Supplemental Table S4. The 70- to 90-nt RNA fraction, which contained mature tRNAs, was initially gel-purified from total RNA using denaturing PAGE. To ligate the 5′-adapter, to the 5′-end of cyto tRNA^HisGUG, 500 ng of the tRNA fraction were incubated with 100 pmol of AS-disrupter in a 4-µL reaction mixture at 90°C for 2 min and subsequently incubated at 37°C. RNA was then added immediately to a ligation reaction mixture (total volume: 10 µL) containing 200 pmol of 5′-adapter, T4 RNA ligase 1 (New England Biolabs), 10% (v/v) DMSO, and 5% PEG8000 and incubated at 37°C for 1 h, followed by an overnight incubation at 4°C. Next, 1 µL of the ligation mixture was subjected to cDNA synthesis with 1 µM of RT primer and SuperScript III (Invitrogen). For TaqMan qPCR quantification, the cDNA product (0.5 µL of the RT mixture) was added to a reaction mixture (total volume: 10 µL) containing 5 µL of qPCR Master Mix (TaKaRa), 0.2 µM each of reverse and forward primers, and 0.1 µM of TaqMan probe. Using a StepOne Plus Real-time PCR machine (Applied Biosystems), the reaction mixture was incubated at 95°C for 20 sec, followed by 40 cycles of 95°C for 1 sec and 65°C for 20 sec.