Figure 5

Detection of 5′ tRNA halves in mouse serum. Northern blot analysis of RNA extracted from U2OS cells cultured in the absence (−) or presence (+) of sodium arsenite (AS), or from 0.4 ml of mouse serum. The blot was hybridized to a ³²P-end-labeled oligonucleotide probe complementary to the 5′ (A) or 3′ end (B) of tRNA-Gly-GCC. The blot was also hybridized to a ³²P-end-labeled oligonucleotide probe complementary to the 5′ (C) or 3′ end (D) of tRNA-Val-CAC. 5′ tRNA halves were also detected in fractionated mouse serum. (E-F) Northern blotting analysis was carried out on RNA extracted from either 0.4 ml of mouse whole serum, from the supernatant (Sup) after ultracentrifugation of 0.4 ml of mouse serum at 110000g, or the ultracentrifugation pellet. The blot was hybridized to a ³²P-end-labeled oligonucleotide probe complementary to the 5′ (E) or 3′ end (F) of tRNA-Gly-GCC. (G) Ultrafiltration indicates a size for tRNA serum particles between 100 and 300 kDa. Samples of 0.2 ml serum mixed with 1.8 ml PBS were subjected to ultrafiltration through Vivaspin 2 columns with 30, 100, and 300 kDa MW cut-offs. Total RNAs were extracted from filtrate (f) and concentrate (c) fractions. Blot was hybridized to ³²P-end-labeled oligonucleotide probes complementary to the 5′ end of tRNA-Gly-GCC. The positions of full length tRNAs and tRNA halves are indicated on the right. DM: decade markers.