Shigematsu Megumi, Honda Shozo, Kirino Yohei
Computational Medicine Center, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA, 19107, USA.
Computational Medicine Center, Sidney Kimmel Medical College, Thomas Jefferson University, 1020 Locust Street, JAH Suite #M77, Philadelphia, PA, 19107, USA.
Methods Mol Biol. 2018;1680:65-73. doi: 10.1007/978-1-4939-7339-2_4.
Cellular RNAs are often expressed as multiple isoforms of complex heterogeneity in both length and terminal sequences. IsomiRs, the isoforms of microRNAs, are such an example. Distinct quantification of each RNA variant is necessary to unravel the biogenesis mechanism and biological significance of heterogenetic RNA expression. Here we describe Dumbbell-PCR (Db-PCR), a TaqMan RT-PCR-based method that distinctively quantifies a specific small RNA variant with single-nucleotide resolution at terminal sequences. Db-PCR enables the quantitative analysis of RNA terminal heterogeneity without performing Next-Generation Sequencing.
细胞RNA通常以长度和末端序列复杂多样的多种异构体形式表达。微小RNA的异构体（isomiRs）就是这样一个例子。对每个RNA变体进行独特的定量分析对于揭示异源RNA表达的生物发生机制和生物学意义至关重要。在这里，我们描述了哑铃状PCR（Db-PCR），这是一种基于TaqMan RT-PCR的方法，能够在末端序列上以单核苷酸分辨率独特地定量分析特定的小RNA变体。Db-PCR无需进行下一代测序就能对RNA末端异质性进行定量分析。
