Quantification of adenosine-to-inosine editing of microRNAs using a conventional method.

In this protocol, I describe a method for measuring the frequency of adenosine-to-inosine RNA editing of primary, precursor and mature forms of specific microRNAs (miRNAs) derived from the same source. The procedure involves reverse transcription (RT)-PCR amplification of regions containing the editing sites followed by subcloning of the PCR products and sequencing. In contrast to deep sequencing, this method does not require any specialized equipment. Pri-miRNAs, which are relatively long primary transcripts, are amplified using a conventional RT-PCR method. Therefore, this method can be adapted for any known RNA-editing sites. In contrast, 3' polyadenylation followed by 5' adaptor ligation is indispensable for amplification of pre-miRNAs and mature miRNAs. The complete protocol takes ∼1 week. I also include details of direct sequence analysis of the PCR products derived from pri-miRNAs as an alternative method. Although it is not as precise as the subcloning method, this procedure enables us to study RNA-editing events of many samples.