Honda Shozo, Shigematsu Megumi, Morichika Keisuke, Telonis Aristeidis G, Kirino Yohei
a Computational Medicine Center ; Sidney Kimmel Medical College ; Thomas Jefferson University ; Philadelphia , PA USA.
RNA Biol. 2015;12(5):501-8. doi: 10.1080/15476286.2015.1031951.
Transfer RNAs (tRNAs) play a central role in translation and also recently appear to have a variety of other functions in biological processes beyond translation. Here we report the development of Four-Leaf clover qRT-PCR (FL-PCR), a convenient PCR-based method, which can specifically quantify individual mature tRNA species. In FL-PCR, T4 RNA ligase 2 specifically ligates a stem-loop adapter to mature tRNAs but not to precursor tRNAs or tRNA fragments. Subsequent TaqMan qRT-PCR amplifies only unmodified regions of the tRNA-adapter ligation products; therefore, FL-PCR quantification is not influenced by tRNA post-transcriptional modifications. FL-PCR has broad applicability for the quantification of various tRNAs in different cell types, and thus provides a much-needed simple method for analyzing tRNA abundance and heterogeneity.
转运RNA（tRNA）在翻译过程中发挥核心作用，并且最近似乎在翻译之外的生物过程中也具有多种其他功能。在此，我们报告了四叶草定量逆转录聚合酶链反应（FL-PCR）的开发，这是一种基于聚合酶链反应的便捷方法，可特异性定量单个成熟tRNA种类。在FL-PCR中，T4 RNA连接酶2特异性地将茎环衔接子连接到成熟tRNA上，而不连接到前体tRNA或tRNA片段上。随后的TaqMan定量逆转录聚合酶链反应仅扩增tRNA-衔接子连接产物的未修饰区域；因此，FL-PCR定量不受tRNA转录后修饰的影响。FL-PCR在定量不同细胞类型中的各种tRNA方面具有广泛的适用性，从而为分析tRNA丰度和异质性提供了一种急需的简单方法。
