Detection and quantification of glycosylated queuosine modified tRNAs by acid denaturing and APB gels

Queuosine and glycosylated queuosine modification and acid denaturing gel/northern blot detection. (A) Structure of the protonated queuosine and glycosylated queuosine base. (B) Schematic of acid denaturing gel and northern blot detection of Q- and glycosylated Q-modified tRNAs. Northern blots can be performed using either ³²P-labeled or 5′ biotin-labeled tRNA probes. (C) Acid denaturing gel and northern blot showing separation of glycosylated Q-modified tRNA^Asp (mQ: mannosyl-Q) and tRNA^Tyr (gQ: galactosyl-Q) from unmodified tRNA (G) using ³²P labeled probes. Asterisk (*) indicates an unknown tRNA^Tyr species. 0Q: total RNA from cells without Q-modification; 100Q: total RNA from cells with full Q-modification. Total RNA was from HEK293T cells.