Antisense sequencing improves the accuracy and precision of A-to-I editing measurements using the peak height ratio method

BACKGROUND: A-to-I RNA editing is found in all phyla of animals and contributes to transcript diversity that may have profound impacts on behavior and physiology. Many transcripts of genes involved in axonal conductance, synaptic transmission and modulation are the targets of A-to-I RNA editing. There are a number of methods to measure the extent of A-to-I RNA editing, but they are generally costly and time consuming. One way to determine the frequency of A-to-I RNA editing is the peak height ratio method, which compares the size of peaks on electropherograms that represent unedited and edited sites. FINDINGS: Sequencing of 4 editing sites of the Dα6 nicotinic acetylcholine receptor subunit with an antisense primer (which uses T/C peaks to measure unedited and edited sites, respectively) showed very accurate and precise measurements of A-to-I RNA editing. The accuracy and precision were excellent for all editing sites, including those edited with high or low frequencies. The frequency of A-to-I RNA editing was comparable to the editing frequency as measured by clone counting from the same sample. Sequencing these same sites with the sense primer (which uses A/G peaks) yielded inaccurate and imprecise measurements. CONCLUSIONS: We have validated and improved the accuracy and precision of the peak height ratio method to measure the frequency of A-to-I RNA editing, and shown that results are primer specific. Thus, the correct sequencing primer must be utilized for the most dependable data. When compared to other methods used to measure the frequency of A-to-I RNA editing, the major benefits of the peak height ratio are that this method is inexpensive, fast, non-labor intensive and easily adaptable to many laboratory and field settings.