Nytran plus membranes

Tissue samples (50–100 mg, flash-frozen in liquid N₂) were homogenized into Qiazol lysis reagent (Qiagen), and RNA was purified according to the manufacturer's directions. RNA was precipitated twice, quantified, and resolved by denaturing polyacrylamide electrophoresis before electrophoretic transfer to Nytran Plus membranes (GE Healthcare) and hybridization with [³²P]-end labeled oligonucleotide probes at 42°C (Li et al. 2000 (link)). tRNA signals detected by phosphorimaging were quantified and normalized to U3 snRNA to compare expression in wild-type and knockout samples. For in vivo labeling, mice (23 wk of age) maintained on a breeder chow diet were fasted overnight and injected i.p. with 0.5 mCi ³²P-orthophosphate (carrier-free) in Tris-buffered saline. After 4 h, the animals were sacrificed, tissues were dissected and freeze-clamped, and total RNA was prepared for electrophoresis on denaturing polyacrylamide gels.

Cerebral hemispheres and livers were harvested and snap-frozen in liquid nitrogen. Of note, one-year-old mice were fed ad libitum for their entire life, while 90-days-old mice used for Northern Blots were fasted for 16 h and refed for 5 h prior to sacrifice and tissue collection in order to stimulate Pol III transcription. Tissues were homogenized in Qiazol lysis reagent (Qiagen). Total RNA was extracted with the miRNeasy kit (Qiagen) and treated with DNAse I (Qiagen) according to the manufacturer’s instructions. RNA quality was assessed on an Agilent 2100 Bioanalyzer and RNA Integrity Numbers (RIN) were routinely above 9. For Northern Blots, RNA samples (7.5ug or 10 ug) were separated by denaturing polyacrylamide gel electrophoresis and transferred to Nytran Plus membranes (GE Healthcare). The resulting blots were sequentially hybridized with [³²P]-end labelled probes detecting precursor tRNA^Ile(TAT), mature tRNA^Leu(AAG) and mature tRNA^Glu(CTC) at 42 °C. Bc1 RNA levels were subsequently detected with a probe mapping to the 5′ portion of the RNA, as previously described[72 (link)]. For n-Tr20, blots were sequentially hybridized with a probe targeting the 3′ trailer sequence of precursor n-Tr20 and a probe targeting both precursor and mature n-Tr20 [37 (link)]. All Pol III transcript levels were quantified and normalized to U3 snRNA levels.