antisense sequencing improves the accuracy and precision of a-to-i editing measurements using the peak height ratio method反义序列改善a-to-i编辑的准确性和精度测量采用峰高比值法.pdfVIP

Rinkevich et al. BMC Research Notes 2012, 5:63 /1756-0500/5/63 TECHNICAL NOTE Open Access Antisense sequencing improves the accuracy and precision of A-to-I editing measurements using the peak height ratio method 1 2 1* Frank D Rinkevich , Peter A Schweitzer and Jeffrey G Scott Abstract 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 Da6 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 ut