Department of Microbiology
and The Center for RNA Biology, The Ohio State University, Columbus, OH
43210-1292, USA.
The primary sequence of all nucleic acids in a cell contain 4 canonical
nucleotides (G, A, T, and C for DNA and G, A, U, and C for RNA).
However, post-transcriptionally, nucleic acids can undergo a number of
chemical modifications, which may change their structure and function.
tRNAs contain the most diverse array of post-transcriptionally added
chemical groups that involve both editing and modification. Because
editing and modification events can serve vital roles in cell function,
it is important to develop techniques that allow for fast and accurate
analysis of these events. This chapter describes the methods used to
purify tRNAs from total native RNA pools and for subsequent analysis of
their edited and modified states using reverse transcriptase-based
approaches. These techniques, in combination with 2D-TLC, allow for the
routine analysis and quantitation of edited and modified nucleotides in a
fast, cost effective manner and without the need for special equipment
such as HPLC or a mass spectrometer. Admittedly, the techniques
described here are only applicable to a subset of post-transcriptional
changes occurring in a tRNA such as C to U and A to I editing as well as
modifications that prevent reverse transcriptase elongation; these have
been highlighted throughout the chapter.
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