Experimental validation of tRF-5s. (A) Sequence alignment of three validated tRF5s with their parental mature tRNAs and Northern probes. The letters in bold indicate the codon sequences. (B) Total RNA from A549 cells, treated with arsenite for 6 h at indicated concentrations was loaded to a denaturing polyacrylamide gel for Northern hybridization using probes indicated in panel A. Untreated cells were used as control. Total RNAs stained with ethidium bromide (EtBr) staining and Northern detection on the 5 S rRNA are shown for equal loading. The positions of tRF-5 and mature tRNA are indicated on the right; molecular size markers are indicated on the left. The blot was exposed for 8 h, 1 day, and 5 days for the detection of tRF5-GluCTC, tRF5-ProTGG, and tRF5-AlaCGC, respectively. Data are representative of three independent experiments. (C) Densitometric analysis of the tRF bands from three Northern blots was performed for 1B, using the histogram function of Adobe Photoshop (San Jose, CA). Basically, the mean tRF intensity was normalized by the corresponding mean intensity 5 S rRNA and expressed as mean ± standard error (SE). For two bands of tRF5-GluCTC in the same treatment, their mean intensity was first calculated, followed by normalization. (D,E) The induction of tRF5-GluCTC, tRF5-ProTGG, and tRF5-AlaCGC were also inducible by arsentite in SAE cells. The Northern blot was done similarly as described in B, while the band intensity was quantified as described in C. Data shown are representative of three independent experiments. * and ** represent P < 0.05 and P < 0.01 respectively, relative to CN oligo-treated cells.