RNA Bioinformatics and High Throughput Analysis, Friedrich Schiller University, Jena, Germany.
Leibniz Institute on Aging-Fritz Lipmann Institute (FLI), Jena, Germany.
Nat Commun. 2024 Jul 12;15(1):5858. doi: 10.1038/s41467-024-49685-0.
Reverse transcription (RT) is a crucial step in most RNA analysis methods. Optimizing protocols for this initial stage is critical for effective target detection, particularly when working with limited input RNA. Several factors, such as the input material quality and reaction conditions, influence RT efficiency. However, the effect of RT primer length on gene detection efficiency remains largely unknown. Thus, we investigate its impact by generating RNA-seq libraries with random RT primers of 6, 12, 18, or 24 nucleotides. To our surprise, the 18mer primer shows superior efficiency in overall transcript detection compared to the commonly used 6mer primer, especially in detecting longer RNA transcripts in complex human tissue samples. This study highlights the critical role of primer length in RT efficiency, which has significant potential to benefit various transcriptomic assays, from basic research to clinical diagnostics, given the central role of RT in RNA-related analyses.
反转录（RT）是大多数 RNA 分析方法中的关键步骤。优化该初始阶段的方案对于有效目标检测至关重要，特别是在处理有限的输入 RNA 时。许多因素，如输入材料质量和反应条件，都会影响 RT 效率。然而，RT 引物长度对基因检测效率的影响在很大程度上仍然未知。因此，我们通过生成具有 6、12、18 或 24 个核苷酸随机 RT 引物的 RNA-seq 文库来研究其影响。令我们惊讶的是，与常用的 6 个核苷酸引物相比，18 个核苷酸引物在整体转录本检测中显示出更高的效率，尤其是在检测复杂人体组织样本中的较长 RNA 转录本时。这项研究强调了引物长度在 RT 效率中的关键作用，鉴于 RT 在 RNA 相关分析中的核心作用，它具有显著潜力使从基础研究到临床诊断等各种转录组学分析受益。
