Modular RCA-CRISPR/Cas12a amplification on a multi-volume SlipChip for ultrafast, single-copy quantification of circRNA and miRNA in ovarian cancer.

IF 5.4 2区 工程技术 Q1 BIOCHEMICAL RESEARCH METHODS
Lab on a Chip Pub Date : 2025-08-28 DOI:10.1039/d5lc00585j
Lingxi Tian, Yan Gao, Yang Lu, Feng Xu, Zirui Feng, Lihan Zi, Zaian Deng, Jun Yang
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引用次数: 0

Abstract

The aberrant expression of RNAs in ovarian cancer (OC) progression highlights their potential as clinical biomarkers. However, rapid and accurate quantification of these RNAs in biosamples remains a significant challenge. In this study, we develop a modular isothermal rolling circle amplification (RCA)-activated Cas12a loop-enhanced (MIRACLE) amplification method for circRNA and miRNA quantification without the need of reverse transcription. In this design, isothermal amplification of modular DNA can be initiated by target-specific RCA primers or miRNAs, with the amplification products subsequently recognized by the Cas12a system to generate measurable signals. When integrated with a multi-volume sliding chip (SlipChip) platform, this MIRACLE method enables portable, rapid and ultra-sensitive quantification of these two types of RNA. Under optimized conditions, this platform exhibits detection limits of 0.125 copies per μL for circRNA and 0.326 copies per μL for miRNA, covering a 5-log dynamic range from 10-1 to 103 copies per μL within 35 min. The platform was validated using OC cell lines and clinical blood samples. It successfully profiled OC RNA biomarkers (hsa_circ_0049101 and hsa-miR-338-3p) and effectively distinguished between early and advanced stages of OC. These results show a strong correlation with RT-qPCR (R2 = 0.953 for circRNA and R2 = 0.947 for miRNA). This work establishes a versatile CRISPR-microfluidic platform for cancer diagnosis. Its modular design allows for adaptation to detect other cancer-related RNA biomarkers, thereby addressing critical needs in precision oncology.

模块化RCA-CRISPR/Cas12a在多体积SlipChip上扩增,用于卵巢癌中circRNA和miRNA的超快速、单拷贝定量。
rna在卵巢癌(OC)进展中的异常表达突出了它们作为临床生物标志物的潜力。然而,在生物样品中快速准确地定量这些rna仍然是一个重大挑战。在本研究中,我们开发了一种模块化等温滚动环扩增(RCA)激活的Cas12a环增强(MIRACLE)扩增方法,用于circRNA和miRNA的定量,而无需逆转录。在本设计中,模块化DNA的等温扩增可以由靶向性RCA引物或mirna启动,扩增产物随后被Cas12a系统识别,产生可测量的信号。当与多体积滑动芯片(SlipChip)平台集成时,这种MIRACLE方法可以实现便携式,快速和超灵敏的定量这两种类型的RNA。在优化条件下,该平台对circRNA的检测限为0.125 copies / μL,对miRNA的检测限为0.326 copies / μL,在35 min内覆盖10-1 ~ 103 copies / μL的5对数动态范围。该平台使用OC细胞系和临床血液样本进行验证。它成功地分析了OC RNA生物标志物(hsa_circ_0049101和hsa-miR-338-3p),并有效地区分了早期和晚期OC。这些结果显示与RT-qPCR有很强的相关性(circRNA R2 = 0.953, miRNA R2 = 0.947)。本工作建立了一个多功能的crispr -微流控癌症诊断平台。其模块化设计允许适应检测其他癌症相关的RNA生物标志物,从而满足精确肿瘤学的关键需求。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Lab on a Chip
Lab on a Chip 工程技术-化学综合
CiteScore
11.10
自引率
8.20%
发文量
434
审稿时长
2.6 months
期刊介绍: Lab on a Chip is the premiere journal that publishes cutting-edge research in the field of miniaturization. By their very nature, microfluidic/nanofluidic/miniaturized systems are at the intersection of disciplines, spanning fundamental research to high-end application, which is reflected by the broad readership of the journal. Lab on a Chip publishes two types of papers on original research: full-length research papers and communications. Papers should demonstrate innovations, which can come from technical advancements or applications addressing pressing needs in globally important areas. The journal also publishes Comments, Reviews, and Perspectives.
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