Guangdong Provincial Key Laboratory of Sensor Technology and Biomedical Instrument, School of Biomedical Engineering, Shenzhen Campus of Sun Yat-sen University, Shenzhen, 518107, China.
Department of Medical Laboratory, Shenzhen People's Hospital, (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, 518020, China.
Biosens Bioelectron. 2025 Jan 1;267:116825. doi: 10.1016/j.bios.2024.116825. Epub 2024 Oct 1.
Circular RNA (circRNA) and microRNA (miRNA) are both non-coding RNAs (ncRNAs) that serve as biomarkers for cancer diagnosis and prognosis. Quantitative detection of these ncRNAs is of particular importance to elucidate the functional mechanisms and evaluate their potential as biomarkers. However, the inherent structures of circRNA and miRNA are different from the mRNA, conventional qRT-PCR is unsuitable for the detection of these ncRNAs. Here, we propose a sensitive method for quantitative detection of circRNA and miRNA using polydisperse droplet digital CRISPR/Cas13a (PddCas13a). It can achieve limits of detection (LOD) as low as ∼10 aM without polymerase-based amplification. To efficiently detect the circRNA and miRNA in real samples, we use a chemically modified crRNA to enhance the stability of crRNA and improve the performance of Cas13a in complex environments containing contaminants. By integrating an extraction-free procedure with PddCas13a, we experimentally demonstrate the applicability of PddCas13a by testing clinical samples. Furthermore, we develop an automated and portable instrument for PddCas13a and verify its applicability for the detection of circRNA and miRNA from exosomes in point-of-care (POC) setting. This is the first report to detect the circRNA and miRNA simultaneously in POC setting. We envision this platform could promote the research of ncRNAs.
环状 RNA（circRNA）和 microRNA（miRNA）都是非编码 RNA（ncRNA），可作为癌症诊断和预后的生物标志物。定量检测这些 ncRNA 对于阐明其功能机制和评估其作为生物标志物的潜力非常重要。然而，circRNA 和 miRNA 的固有结构与 mRNA 不同，常规 qRT-PCR 不适合检测这些 ncRNA。在这里，我们提出了一种使用多分散液滴数字 CRISPR/Cas13a（PddCas13a）定量检测 circRNA 和 miRNA 的灵敏方法。它可以在没有聚合酶扩增的情况下实现低至约 10 aM 的检测限（LOD）。为了有效地检测真实样本中的 circRNA 和 miRNA，我们使用化学修饰的 crRNA 来增强 crRNA 的稳定性，并提高 Cas13a 在含有污染物的复杂环境中的性能。通过将无提取程序与 PddCas13a 集成，我们通过测试临床样本实验证明了 PddCas13a 的适用性。此外，我们开发了用于 PddCas13a 的自动化和便携式仪器，并验证了其在即时护理（POC）环境中检测外泌体中 circRNA 和 miRNA 的适用性。这是第一个在 POC 环境中同时检测 circRNA 和 miRNA 的报告。我们设想这个平台可以促进 ncRNA 的研究。
