Biochemical Identification of A-to-I RNA Editing Sites by the Inosine Chemical Erasing (ICE) Method

Adenosine-to-inosine (A-to-I) RNA editing is a biologically important posttranscriptional processing event involved in the transcriptome diversification. The most conventional method of editing site identification is to compare the cDNA sequence with its corresponding genomic sequence; however, using this method, it is difficult to discriminate between guanosine residue that originated from inosine and errors or noise in the sequencing chromatograms. To address this issue, we developed the inosine chemical erasing (ICE) method to identify inosines in RNA strands utilizing inosine cyanoethylation and reverse transcription PCR. Since this method requires only a limited quantity of total RNA, it can be used in the genome-wide profiling of A-to-I editing sites in tissues and cells from various organisms, including clinical specimens.