TY - JOUR
T1 - Single-Tube Reaction Using Perfluorocarbons: A Prerequisite Step Leading to the Whole-Slide In Situ Technique on Histopathological Slides
AU - Chen, Yi-Chang
AU - Teng, Tsung-Han
AU - Tsai, Jane S. -C.
AU - Huang, Hsien-Da
AU - Chang, Yih-Leong
AU - Liang, Cher-Wei
PY - 2016/1/23
Y1 - 2016/1/23
N2 - 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.
AB - 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.
KW - MESSENGER-RNA; PCR; IMMUNOHISTOCHEMISTRY; HETEROGENEITY; AMPLIFICATION
U2 - 10.1371/journal.pone.0158018
DO - 10.1371/journal.pone.0158018
M3 - Article
VL - 11
JO - PLoS ONE
JF - PLoS ONE
SN - 1932-6203
IS - 6
M1 - e0158018
ER -