To exclude that the qPCR data was biased due to impaired RT of fully modified tRNAs, we utilized Northern blotting, the most established method for tRNA assessment, generating probes to interrogate representative unique isoforms for pre-tRNA for Tyrosine Chr14:tRNA19 and Isoleucine Chr19:tRNA10, as well as to detect all isoacceptor (pan: all isoacceptor, i.e. same anticodon) for pre-tRNA and mature tRNA for Tyrosine, Isoleucine and Leucine. Most probes showed some evidence for expression in both fibroblasts and iNeurons with the exception of pre-tRNA for Chr14:tRNA19-TyrGTA, which was detectable only in iNeurons, run in duplicate to avoid sample loading variability. We identified both accumulation of pre-tRNAs as well as depletion of mature tRNAs specifically in patient iNeurons. Pre-tRNA Chr14:tRNA19-TyrGTA, pre-tRNA Chr19:tRNA10-IleTAT, as well as pre-tRNA IleTAT (pan) and pre-tRNA LeuCAA (pan) showed stronger bands in affected vs. unaffected iNeurons (Figure 4D). We also noted an accumulation of Chr19.tRNA10-IleTAT intron (data not shown). Correspondingly, there was a reduction in the amounts of mature tRNAs for Isoleucine and Leucine for mature tRNA IleTAT (pan) and mature tRNA LeuCAA (pan). These results were quantified as
