O'Keeffe Piper, Nouri Yasmin, Saw Hui Shi, Moore Zachery, Baldwin Tracey M, Olechnowicz Sam W Z, Jabbari Jafar S, Squire David McG, Leslie Stephen, Wang Changqing, You Yupei, Ritchie Matthew E, Cross Ryan S, Jenkins Misty R, Audiger Cindy, Naik Shalin H, Whittle James R, Freytag Saskia, Best Sarah A, Hickey Peter F, Amann-Zalcenstein Daniela, Bowden Rory, Brown Daniel V
The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade VIC, Melbourne, VIC, 3052, Australia.
Department of Medical Biology, University of Melbourne, Parkville, VIC, 3010, Australia.
Sci Rep. 2025 May 2;15(1):15385. doi: 10.1038/s41598-025-98282-8.
RNA sequencing (RNA-seq) is widely used in biomedical research, advancing our understanding of gene expression across biological systems. Traditional methods require upstream RNA extraction from biological inputs, adding time and expense to workflows. We developed TIRE-seq (Turbocapture Integrated RNA Expression Sequencing) to address these challenges. TIRE-seq integrates mRNA purification directly into library preparation, eliminating the need for a separate extraction step. This streamlined approach reduces turnaround time, minimizes sample loss, and improves data quality. A comparative study with the widely used Prime-seq protocol demonstrates TIRE-seq's superior sequencing efficiency with crude cell lysates as inputs. TIRE-seq's utility was demonstrated across three biological applications. It captured transcriptional changes in stimulated human T cells, revealing activation-associated gene expression profiles. It also identified key genes driving murine dendritic cell differentiation, providing insights into lineage commitment. Lastly, TIRE-seq analyzed the dose-response and time-course effects of temozolomide on patient-derived neurospheres, identifying differentially expressed genes and enriched pathways linked to the drug's mechanism of action. With its simplified workflow and high sequencing efficiency, TIRE-seq offers a cost-effective solution for large-scale gene expression studies across diverse biological systems.
RNA测序（RNA-seq）在生物医学研究中被广泛应用，加深了我们对跨生物系统基因表达的理解。传统方法需要从生物样本中进行上游RNA提取，这增加了工作流程的时间和成本。我们开发了TIRE-seq（TurboCapture集成RNA表达测序）来应对这些挑战。TIRE-seq将mRNA纯化直接整合到文库制备中，无需单独的提取步骤。这种简化的方法减少了周转时间，最大限度地减少了样本损失，并提高了数据质量。与广泛使用的Prime-seq方案的比较研究表明，以粗细胞裂解物为输入时，TIRE-seq具有更高的测序效率。TIRE-seq的实用性在三个生物学应用中得到了证明。它捕获了刺激的人类T细胞中的转录变化，揭示了与激活相关的基因表达谱。它还确定了驱动小鼠树突状细胞分化的关键基因，为细胞谱系定向提供了见解。最后，TIRE-seq分析了替莫唑胺对患者来源的神经球的剂量反应和时间进程效应，确定了差异表达基因和与药物作用机制相关的富集途径。凭借其简化的工作流程和高测序效率，TIRE-seq为跨多种生物系统的大规模基因表达研究提供了一种经济高效的解决方案。
