| Info. | Vol.13 - No.2 (2019.06.20) |
|---|---|
| Title | Development of Hydrogel Microparticle based RT-qPCR for Advanced Detection of BCR-ABL1 Transcripts |
| Authors | Jung Min Kim1,2,†, Won Jin Kim1,3,†, Mi Yeon Kim1,2, Kwang Pyo Kim3,4,5,6, Sang Jun Sim2 & Sang Kyung Kim1,7,* |
| Institutions | 1Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), 5, Hwarang-ro 14-gil, Seongbuk-gu, Seoul, 02792, Korea 2Department of Chemical and Biological Engineering, Korea University, Seoul, Korea 3Department of Applied Chemistry, College of Applied Science, Kyung Hee University, Yongin, Korea 4Korea Department of Applied Chemistry, Institute of Natural Science 5Global Center for Pharmaceutical Ingredient Materials, Kyung Hee University, Korea 6Department of Biomedical Science and Technology, Kyung Hee Medical Science Research Institute, Kyung Hee University, Seoul, Korea 7Department of Biomedical Engineering, University of Science and Technology (UST), 217, Gajeong-ro, Yuseong-gu, Daejeon, 34113, Korea †These authors contributed equally to this work *Correspondence and requests for materials should be addressed to S.K. Kim (sangk@kist.re.kr) |
| 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). |
| Keyword | N Real-time PCR, RT-qPCR, Hydrogel microparticle, TaqMan probe, Chronic myeloid leukemia, BCR-ABL |
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