Comparing methods of detection and quantitation of RNA editing of rat glycine receptor alpha3(P185L)

Background: Recently, it has become evident that RNA editing-related changes are important in the modulation of neuronal information processing. Alternatively edited transcripts, when meagerly present, are hard to detect. Significant functional consequences may result, however, from small differences in editing efficiency. Moreover, it is difficult to evaluate the ratio of edited transcripts. The glycine receptor alpha3 subunit (GlyR alpha3) is expressed in the spinal cord, and transcripts of GlyR alpha3 are susceptible to RNA editing. The physiological role of this editing is still unclear. To analyze changes in RNA editing in various animal models, we need reliable and practical ways to detect and quantitate GlyR alpha3 RNA editing. Results: We identified and assessed different ways of detecting edited RNA transcripts, including direct sequencing, denaturing high performance chromatography (DHPLC), allele-specific real-time PCR with TaqMan probes, and PCR with allele-specific primers. Using PCR with allele-specific primers on standard PCR products for edited and nonedited GlyR alpha3, we were able to detect as little as a 0.5% incidence of edited transcripts. We were able to detect a 5% incidence of RNA editing using direct sequencing and 2% using DHPLC. We could accurately determine the ratio of edited to non-edited RNA using DHPLC, direct sequencing, and allele-specific real-time PCR with TaqMan probes. Conclusion: We demonstrated exact and sensitive methods of detecting RNA editing. In prepared samples, we showed means of quantitating the incidence of editing of a particular site. The demonstrated methodologies should be very useful when extended to the evaluation of other types of RNA editing and single base mutations.