Rapid quantification of microRNA-375 through one-pot primer-generating rolling circle amplification,Analyst

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Rapid quantification of microRNA-375 through one-pot primer-generating rolling circle amplification
Analyst ( IF 3.6 ) Pub Date : 2022-06-07 , DOI: 10.1039/d2an00263a
Lucas D Smith _{1,

2} , Siva Nalla _{1,

2} , Chia-Wei Kuo _{1,

2} , Manish Kohli _{3,

4} , Andrew M Smith _{1,

2,

4,

5,

6,

7}

Affiliation

A recent surge of interest in microRNA has been driven by its discovery as a circulating biomarker of disease, with many diagnostic test platforms currently under development. Alternatives to widely used microRNA quantification methods such as quantitative reverse transcriptase PCR (qRT-PCR) are needed for use in portable and point-of-care devices which are incompatible with complex sample processing workflows and thermal cycling. Rolling circle amplification (RCA) is a one-pot assay technique which directly amplifies nucleic acids using sequence-specific microRNA priming to initiate a single-step isothermal reaction that is compatible with simple devices. Sensitivity remains a limitation of RCA methods, however, and detection limits do not typically reach the femtomolar level in which microRNA targets are present in blood. RCA assays have previously been improved by digestion of the amplification products using a nicking endonuclease to exponentially generate new reaction primers. Here we describe how a ligation-free version of this technique performed in a single tube can be used to improve the limit of detection for microRNA-375, an important blood biomarker for prostate cancer. Endonuclease addition changes a linear process into an exponential amplification reaction which results in a 61-fold improvement of the limit of detection (5.9 fM), a dynamic range wider than 5-log(10), and a shorter reaction time. By eliminating the need for microRNA reverse transcription and thermal cycling, this single-step one-pot method provides a more rapid and simplified alternative to qRT-PCR for ultrasensitive microRNA quantification in blood extracts.

中文翻译：

通过一锅引物生成滚环扩增快速定量 microRNA-375

最近人们对 microRNA 的兴趣激增，是由于它作为疾病的循环生物标志物的发现，目前许多诊断测试平台正在开发中。需要替代广泛使用的 microRNA 定量方法，例如定量逆转录酶 PCR (qRT-PCR)，以用于与复杂的样品处理工作流程和热循环不兼容的便携式和护理点设备。滚环扩增 (RCA) 是一种一锅测定技术，它使用序列特异性 microRNA 引发直接扩增核酸，以启动与简单设备兼容的单步等温反应。然而，灵敏度仍然是 RCA 方法的一个限制，并且检测限通常不会达到血液中存在 microRNA 靶点的飞摩尔水平。之前，通过使用切口核酸内切酶消化扩增产物，以指数方式生成新的反应引物，对 RCA 测定进行了改进。在这里，我们描述了如何使用在单管中执行的该技术的无连接版本来提高 microRNA-375（前列腺癌的重要血液生物标志物）的检测限。添加核酸内切酶将线性过程转变为指数扩增反应，从而使检测限 (5.9 fM) 提高 61 倍，动态范围比 5-log(10) 更宽，并且反应时间更短。通过消除对 microRNA 逆转录和热循环的需要，这种单步一锅法为 qRT-PCR 提供了一种更快速、更简化的替代方案，用于血液提取物中超灵敏的 microRNA 定量。

更新日期：2022-06-07

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