Abstract
Reverse transcription – quantitative polymerase chain reaction (RT-qPCR) is conventionally used method to analyze oncogenes, infected DNAs, and mutated tumor associated genes. However it is hard to detect rare genetic targets since the disturbance of undesired amplification often overrides the reaction of very few targets in a myriad of interfering genes. To solve the limitation, we developed primerimmobilized network (PIN) probe RT-qPCR which can detect target RNA with high selectivity and sensitivity. To conduct PIN probe RT-qPCR with high efficiency, design of probe, concentration of immobilized probe and qPCR condition were optimized. The LOD of PIN probe RT-qPCR was 40pg per particle with 89.2% efficiency. When extremely low concentration of RNA was used, result of PIN probe RTqPCR was showed “on/off” signal. Also, target was confirmed only in the “on” particle. The interference effects by non-target PCR products were minimized by target-capturing and washing process in the RT. Using PIN probe RT-qPCR, we successfully detected target RNA in a sample which has a ratio of 1:100,000 (positive RNA : negative RNA).
Original language | English |
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Pages (from-to) | 182-190 |
Number of pages | 9 |
Journal | Biochip Journal |
Volume | 13 |
Issue number | 2 |
DOIs | |
Publication status | Published - 2019 Jun 1 |
Bibliographical note
Funding Information:Acknowledgements This work was supported by the R & D Convergence Pr ogram of National Research Council of Science & Technology (NST) of Republic of Korea (Grant no. CAP-16-02-KIST), and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (Grant no. 2015R1A2A1A10055994). Additionally, this work also was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) & funded by the Korean government (MSIP & MOHW) (No. 2016M3A9B6918639).
Publisher Copyright:
© 2018, The Korean BioChip Society and Springer.
Keywords
- BCR-ABL
- Chronic myeloid leukemia
- Hydrogel microparticle
- N Real-time PCR
- RT-qPCR
- TaqMan probe
ASJC Scopus subject areas
- Biotechnology
- Bioengineering
- Biomedical Engineering
- Electrical and Electronic Engineering