Open AccessSingle-Tube Reaction Using Perfluorocarbons: A Prerequisite Step Leading to the Whole-Slide In Situ Technique on Histopathological Slides
Author(s) -
YiChang Chen,
Tsung-Han Teng,
Jane Tsai,
Hsien-Da Huang,
Yih-Leong Chang,
CherWei Liang
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0158018
Subject(s) - multiplex , lysis buffer , in situ , tube (container) , buffer (optical fiber) , throughput , nucleic acid , lysis , polymerase chain reaction , microemulsion , chromatography , materials science , chemistry , biomedical engineering , nanotechnology , computer science , biology , bioinformatics , biochemistry , medicine , telecommunications , pulmonary surfactant , organic chemistry , gene , composite material , wireless
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.