High-throughput RNA sequencing (RNA-seq) has revolutionized our understanding of gene expression. Widely used RNA-seq methods start with adaptor ligation and cDNA synthesis of biological RNA samples followed by PCR amplification to generate sequencing libraries. These standard methods work well for most cellular RNAs such as mRNA, long noncoding RNA, microRNA, or fragments derived from rRNA, small nuclear RNA and small nucleolar RNA. tRNA is the only class of small cellular RNA for which the standard sequencing methods cannot yet be applied efficiently and quantitatively, although attempts have been made. Significant obstacles for the sequencing of tRNA include the presence of numerous post-transcriptional modifications and its stable and extensive secondary structure, which interfere with cDNA synthesis and adaptor ligation. tRNAs are essential for cells, and their synthesis is under stringent cellular control. Recent evidence shows that tRNA expression and mutations are associated with various diseases such as neurological pathologies and cancer development. The lack of efficient and quantitative tRNA sequencing methods has hindered biological studies of tRNA.
Researchers at the University of Chicago applied two strategies to eliminate or substantially reduce the obstacles of tRNA modification and structure for efficient and quantitative tRNA sequencing.
(a) Schematic representation. (b) Demethylation efficiency for total tRNA as measured by triple quadrupole liquid chromatography–mass spectrometry. (c) RT reaction for both purified tRNA and total RNA as template with (+) or without (−) demethylase treatment. The blue line shows the gel region excised for library construction.