title: Single-Tube Reaction Using Perfluorocarbons: A Prerequisite Step Leading to the Whole-Slide In Situ Technique on Histopathological Slides.
abstract: 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.
authors.affiliation: Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan.|Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu, Taiwan.
authors.forename: Yi-Chang
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu, Taiwan.
authors.forename: Tsung-Han
authors.initials: TH
authors.lastname: Teng
authors.affiliation: Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan.
authors.forename: Jane S-C
authors.initials: JS
authors.lastname: Tsai
authors.affiliation: Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu, Taiwan.
authors.forename: Hsien-Da
authors.initials: HD
authors.lastname: Huang
authors.affiliation: Department and Graduate Institute of Pathology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Yih-Leong
authors.initials: YL
authors.lastname: Chang
authors.affiliation: Department and Graduate Institute of Pathology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Cher-Wei
authors.identifier: http://orcid.org/0000-0001-8903-6082
authors.initials: CW
authors.lastname: Liang
mesh_terms: D000818:Animals; D005466:Fluorocarbons; D006801:Humans; D051379:Mice; D060885:Multiplex Polymerase Chain Reaction; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D005466:Fluorocarbons