Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, Ohio 45221-0172, United States.
Anal Chem. 2012 Oct 16;84(20):8607-13. doi: 10.1021/ac301638c. Epub 2012 Oct 3.
Here, we describe a method for the comparative analysis of ribonucleic acids (RNAs). This method allows sequence or modification information from a previously uncharacterized RNA to be obtained by direct comparison with a reference RNA, whose sequence or modification information is known. This simple and rapid method is enabled by the differential labeling of two RNA samples. One sample, the reference RNA, is labeled with (16)O during enzymatic digestion. The second sample, the candidate or unknown RNA, is labeled with (18)O. By combining the two digests, digestion products that share the same sequence or post-transcriptional modification(s) between the reference and candidate will appear as doublets separated by 2 Da. Sequence or modification differences between the two will generate singlets that can be further characterized to identify how the candidate sequence differs from the reference. We illustrate the application of this approach for sequencing individual RNAs and demonstrate how this method can be used to identify sequence-specific differences in RNA modification. This comparative analysis of RNA digests (CARD) approach is scalable to multiple candidate RNAs using one or multiple reference RNAs and is compatible with existing methods for quantitative analysis of RNAs.
在这里，我们描述了一种用于分析核糖核酸（RNAs）的比较方法。这种方法可以通过与参考 RNA 的直接比较，获得以前未被表征的 RNA 的序列或修饰信息，而参考 RNA 的序列或修饰信息是已知的。这种简单快速的方法得益于两种 RNA 样本的差异标记。一个样本，即参考 RNA，在酶消化过程中用（16）O 标记。第二个样本，即候选或未知 RNA，用（18）O 标记。将两个消化产物混合后，参考和候选之间具有相同序列或转录后修饰的消化产物将以相差 2 Da 的双峰形式出现。两者之间的序列或修饰差异将产生单峰，可以进一步对其进行表征，以确定候选序列与参考序列的差异。我们展示了该方法在单个 RNA 测序中的应用，并演示了如何使用该方法鉴定 RNA 修饰的序列特异性差异。这种 RNA 消化物的比较分析（CARD）方法可以使用一个或多个参考 RNA 扩展到多个候选 RNA，并且与 RNA 定量分析的现有方法兼容。
