Methods for the Detection of Adenosine-to-Inosine Editing Events in Cellular RNA

Modification of RNA is essential for properly expressing the repertoire of RNA transcripts necessary for both cell type and developmental specific functions. RNA modifications serve to dynamically re-wire and fine-tune the genetic information carried by an invariable genome. One important type of RNA modification is RNA editing and the most common and well-studied type of RNA editing is the hydrolytic deamination of adenosine to inosine. Inosine is a biological mimic of guanosine; therefore, when RNA is reverse transcribed, inosine is recognized as guanosine by the reverse transcriptase and a cytidine is incorporated into the complementary DNA (cDNA) strand. During PCR amplification, guanosines pair with the newly incorporated cytidines. As a result, the adenosine-to-inosine (A-to-I) editing events are recognized as adenosine to guanosine changes when comparing the sequences of the genomic DNA to the cDNA. This chapter describes the methods for extracting endogenous RNA for subsequent analyses of A-to-I RNA editing using reverse transcriptase-based approaches. We discuss techniques for the detection of A-to-I RNA editing events in messenger RNA (mRNA), including analyzing editing levels at specific adenosines within the total pool of mRNA versus analyzing editing patterns that occur in individual transcripts and a method for detecting editing events across the entire transcriptome. The detection of RNA editing events and editing levels can be used to better understand normal biological processes and disease states.