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PLoS One. 2016 Jun 23;11(6):e0158018. doi: 10.1371/journal.pone.0158018. eCollection 2016.

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

Author information

1
Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan.
2
Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu, Taiwan.
3
Department and Graduate Institute of Pathology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan.

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.

PMID:
27336363
PMCID:
PMC4919083
DOI:
10.1371/journal.pone.0158018
[Indexed for MEDLINE]
Free PMC Article

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