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Single-Tube Reaction Using Perfluorocarbons: A Prerequisite Step Leading to the Whole-Slide In Situ Technique on Histopathological Slides

机译:使用全氟化碳的单管反应:在组织病理学玻片上形成全玻片原位技术的必要步骤

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摘要

Developing a robust, novel method for performing multiple reactions in a single tube is not only time- and cost-saving but also critical for future high-throughput whole-slide in situ techniques on diseased tissues. In this study, we introduce the use of perfluorocarbons and compound-coated magnetic particles to create pseudochambers in a single tube, allowing different reactions to be performed in different phases. Perfluorocarbons also serve as cell lysis buffer and polymerase chain reaction (PCR) buffer owing to their highly penetrating, repellent and emulsifiable properties. Using this method, nucleic acids can be isolated and purified from various sample types and sizes, followed by PCR, real-time PCR, or multiplex PCR in the same tube. No incubation or enzyme digesting time is needed and the risk of cross-contamination is reduced. Tests can be performed in microemulsions (water-in-oil droplets) containing sequence-specific captures and probes for further high-throughput detection. We present a simple, quick, and robust procedure as a prerequisite step to future high-throughput in situ techniques.
机译:开发一种健壮,新颖的方法以在单个试管中进行多个反应不仅节省时间和成本,而且对于将来在患病组织上的高通量全玻片原位技术至关重要。在这项研究中,我们介绍了使用全氟化碳和涂有化合物的磁性颗粒在单个管中创建假腔室,从而允许在不同阶段进行不同的反应。由于全氟化碳具有高渗透性,驱避性和乳化性,它们还可用作细胞裂解缓冲液和聚合酶链反应(PCR)缓冲液。使用这种方法,可以从各种样品类型和大小中分离和纯化核酸,然后在同一试管中进行PCR,实时PCR或多重PCR。无需孵育或酶消化时间,并且减少了交叉污染的风险。可以在含有序列特异性捕获物和探针的微乳液(油包水液滴)中进行测试,以进行进一步的高通量检测。我们提出了一个简单,快速和强大的过程,作为未来高通量现场技术的前提步骤。

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