MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing, China.
Institute for Precision Medicine, Tsinghua University, Beijing, China.
Clin Transl Med. 2024 Jul;14(7):e1760. doi: 10.1002/ctm2.1760.
Cell-free long RNAs in human plasma and extracellular vesicles (EVs) have shown promise as biomarkers in liquid biopsy, despite their fragmented nature.
To investigate these fragmented cell-free RNAs (cfRNAs), we developed a cost-effective cfRNA sequencing method called DETECTOR-seq (depletion-assisted multiplexed cell-free total RNA sequencing). DETECTOR-seq utilised a meticulously tailored set of customised guide RNAs to remove large amounts of unwanted RNAs (i.e., fragmented ribosomal and mitochondrial RNAs) in human plasma. Early barcoding strategy was implemented to reduce costs and minimise plasma requirements.
Using DETECTOR-seq, we conducted a comprehensive analysis of cell-free transcriptomes in both whole human plasma and EVs. Our analysis revealed discernible distributions of RNA types in plasma and EVs. Plasma exhibited pronounced enrichment in structured circular RNAs, tRNAs, Y RNAs and viral RNAs, while EVs showed enrichment in messenger RNAs (mRNAs) and signal recognition particle RNAs (srpRNAs). Functional pathway analysis highlighted RNA splicing-related ribonucleoproteins (RNPs) and antimicrobial humoral response genes in plasma, while EVs demonstrated enrichment in transcriptional activity, cell migration and antigen receptor-mediated immune signals. Our study indicates the comparable potential of cfRNAs from whole plasma and EVs in distinguishing cancer patients (i.e., colorectal and lung cancer) from healthy donors. And microbial cfRNAs in plasma showed potential in classifying specific cancer types.
Our comprehensive analysis of total and EV cfRNAs in paired plasma samples provides valuable insights for determining the need for EV purification in cfRNA-based studies. We envision the cost effectiveness and efficiency of DETECTOR-seq will empower transcriptome-wide investigations in the fields of cfRNAs and liquid biopsy.
DETECTOR-seq (depletion-assisted multiplexed cell-free total RNA sequencing) enabled efficient and specific depletion of sequences derived from fragmented ribosomal and mitochondrial RNAs in plasma. Distinct human and microbial cell-free RNA (cfRNA) signatures in whole Plasma versus extracellular vesicles (EVs) were revealed. Both Plasma and EV cfRNAs were capable of distinguishing cancer patients from normal individuals, while microbial RNAs in Plasma cfRNAs enabled better classification of cancer types than EV cfRNAs.
尽管游离于细胞外的长 RNA（cfRNA）在其碎片化方面存在固有问题，但在液体活检中，作为生物标志物，cfRNA 在人血浆和细胞外囊泡（EVs）中显示出一定的应用前景。
为了研究这些碎片化的游离 RNA（cfRNA），我们开发了一种具有成本效益的 cfRNA 测序方法，称为 DETECTOR-seq（耗竭辅助的多路复用游离总 RNA 测序）。DETECTOR-seq 利用了一套经过精心设计的定制 guide RNA，以去除人血浆中大量不需要的 RNA（即碎片化的核糖体和线粒体 RNA）。早期的条形码策略被实施以降低成本和最小化血浆需求。
使用 DETECTOR-seq，我们对全血浆和 EV 中的游离转录组进行了全面分析。我们的分析揭示了血浆和 EV 中 RNA 类型的可区分分布。血浆中明显富集了结构环状 RNA、tRNA、Y RNA 和病毒 RNA，而 EV 中则富集了信使 RNA（mRNA）和信号识别颗粒 RNA（srpRNA）。功能途径分析突出了 RNA 剪接相关的核糖核蛋白（RNP）和抗微生物体液反应基因在血浆中的作用，而 EV 则表现出转录活性、细胞迁移和抗原受体介导的免疫信号的富集。我们的研究表明，来自全血浆和 EV 的 cfRNA 具有区分癌症患者（即结直肠癌和肺癌）和健康供体的潜力。此外，血浆中的微生物 cfRNA 具有分类特定癌症类型的潜力。
我们对配对血浆样本中的总 cfRNA 和 EV cfRNA 进行了全面分析，为确定在基于 cfRNA 的研究中是否需要 EV 纯化提供了有价值的见解。我们预计 DETECTOR-seq 的成本效益和效率将为 cfRNA 和液体活检领域的全转录组研究提供支持。
DETECTOR-seq（耗竭辅助的多路复用游离总 RNA 测序）能够有效地特异性耗竭来自血浆中碎片化核糖体和线粒体 RNA 的序列。全血浆与细胞外囊泡（EVs）之间的人类和微生物游离 RNA（cfRNA）特征明显不同。血浆和 EV cfRNA 均能区分癌症患者和正常个体，而血浆 cfRNA 中的微生物 RNA 比 EV cfRNA 更能准确地对癌症类型进行分类。
