Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba, Japan.
Biological Data Science Division, Research Center for Advanced Science and Technology (RCAST), University of Tokyo, Tokyo, Japan.
Methods Mol Biol. 2021;2181:113-148. doi: 10.1007/978-1-0716-0787-9_8.
RNA editing of adenosines to inosines contributes to a wide range of biological processes by regulating gene expression post-transcriptionally. To understand the effect, accurate mapping of inosines is necessary. The most conventional method to identify an editing site is to compare the cDNA sequence with its corresponding genomic sequence. However, this method has a high false discovery rate because guanosine signals, due to experimental errors or noise in the obtained sequences, contaminate genuine inosine signals detected as guanosine. To ensure high accuracy, we developed the Inosine Chemical Erasing (ICE) method to accurately and biochemically identify inosines in RNA strands utilizing inosine cyanoethylation and reverse transcription-PCR. Furthermore, we applied this technique to next-generation sequencing technology, called ICE-seq, to conduct an unbiased genome-wide screening of A-to-I editing sites in the transcriptome.
腺嘌呤核苷到肌苷核苷的 RNA 编辑通过转录后调控基因表达，从而促进了广泛的生物学过程。为了了解其影响，需要准确地对肌苷核苷进行定位。最传统的识别编辑位点的方法是将 cDNA 序列与其相应的基因组序列进行比较。然而，由于在获得的序列中存在实验误差或噪声，鸟嘌呤信号会污染作为鸟嘌呤检测到的真正肌苷核苷信号，因此这种方法的假阳性率很高。为了确保高准确性，我们开发了肌苷核苷化学消除（ICE）方法，利用肌苷核苷氰乙基化和反转录-PCR，准确地对 RNA 链中的肌苷核苷进行生物化学鉴定。此外，我们将该技术应用于下一代测序技术，称为 ICE-seq，对转录组中的 A 到 I 编辑位点进行了无偏基因组范围的筛选。
