Single- Tube reaction using perfluorocarbons: A prerequisite step leading to the whole- slide in situ technique on histopathological slides
Journal
PLoS ONE
Journal Volume
11
Journal Issue
6
Pages
e0158018
Date Issued
2016
Author(s)
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. ? 2016 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Other Subjects
fluorocarbon; animal tissue; Article; chemical procedures; chemical reaction; contamination; controlled study; cross contamination; cytolysis; Dengue virus; enzyme digesting time; gene sequence; histopathology; incubation time; magnetic particle; material state; microemulsion; mouse; multiplex polymerase chain reaction; nonhuman; real time polymerase chain reaction; RNA isolation; single tube reaction; time; whole slide in situ technique; animal; human; nucleic acid amplification; polymerase chain reaction; procedures; Animals; Fluorocarbons; Humans; Mice; Multiplex Polymerase Chain Reaction; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction
Publisher
Public Library of Science
Type
journal article