FIGURE 4.

Quantitative detection of γ-toxin cleavage of mcm5s2u-containing tRNAs using qRT-PCR. (A) Schematic of qRT-PCR assay for detecting tRNA cleavage by γ-toxin. The location of PCR primers for amplification of full-length tRNA-Glu-UUC is denoted by half-arrows. (B) Real-time PCR analysis of cDNA generated from reverse transcription of total RNA isolated from the indicated yeast strain or human cells that were either untreated or pretreated with γ-toxin. The total amount of RNA was normalized using 25S rRNA (yeast) or 5.8S (human) and expressed relative to the untreated total RNA sample of each organism. All assays were performed in triplicate on multiple independent samples and repeated greater than 3× to ensure reproducibility.