Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, United States.
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, United States; Department of Chemistry, Yale University, New Haven, CT, United States; Howard Hughes Medical Institute, Chevy Chase, MD, United States.
Methods Enzymol. 2023;691:3-15. doi: 10.1016/bs.mie.2023.07.002. Epub 2023 Jul 27.
RNA molecules play important roles in numerous normal cellular processes and disease states, from protein coding to gene regulation. RT-PCR, applying the power of polymerase chain reaction (PCR) to RNA by coupling reverse transcription with PCR, is one of the most important techniques to characterize RNA transcripts and monitor gene expression. The ability to analyze full-length RNA transcripts and detect their expression is critical to decipher their biological functions. However, due to the low processivity of retroviral reverse transcriptases (RTs), we can only monitor a small fraction of long RNA transcripts, especially those containing stable secondary and tertiary structures. The full-length sequences can only be deduced by computational analysis, which is often misleading. Group II intron-encoded RTs are a new type of RT enzymes. They have evolved specialized structural elements that unwind template structures and maintain close contact with the RNA template. Therefore, group II intron-encoded RTs are more processive than the retroviral RTs. The discovery, optimization and deployment of processive group II intron RTs provide us the opportunity to analyze RNA transcripts with single molecule resolution. MarathonRT, the most processive group II intron RT, has been extensively optimized for processive reverse transcription. In this chapter, we use MarathonRT to deliver a general protocol for long amplicon generation by RT-PCR, and also provide guidance for troubleshooting and further optimization.
RNA 分子在许多正常的细胞过程和疾病状态中发挥着重要作用，从蛋白质编码到基因调控。RT-PCR 通过将逆转录与 PCR 相结合，利用聚合酶链式反应（PCR）的强大功能来研究 RNA 转录物并监测基因表达，这是一种非常重要的技术。分析全长 RNA 转录物并检测其表达的能力对于破译它们的生物学功能至关重要。然而，由于逆转录病毒逆转录酶（RTs）的低延伸性，我们只能监测一小部分长 RNA 转录物，特别是那些含有稳定的二级和三级结构的转录物。只有通过计算分析才能推断出全长序列，但这往往会产生误导。
内含子编码的 RT 是一种新型的 RT 酶。它们已经进化出专门的结构元件，可以解开模板结构并与 RNA 模板保持紧密接触。因此，内含子编码的 RT 比逆转录病毒 RT 更具延伸性。具有延伸性的内含子 RT 的发现、优化和应用为我们提供了以单分子分辨率分析 RNA 转录物的机会。MarathonRT 是最具延伸性的内含子 RT，已经被广泛优化以进行延伸性逆转录。在这一章中，我们使用 MarathonRT 提供了一种通过 RT-PCR 生成长扩增子的通用方案，并为故障排除和进一步优化提供了指导。
