Development of Hydrogel Microparticle based RT-qPCR for Advanced Detection of BCR-ABL1 Transcripts

Jung Min Kim; Won Jin Kim; Mi Yeon Kim; Kwang Pyo Kim; Sang Jun Sim; Sang Kyung Kim

doi:10.1007/s13206-018-3209-9

Development of Hydrogel Microparticle based RT-qPCR for Advanced Detection of BCR-ABL1 Transcripts

Jung Min Kim, Won Jin Kim, Mi Yeon Kim, Kwang Pyo Kim, Sang Jun Sim, Sang Kyung Kim

Research output: Contribution to journal › Article › peer-review

5 Citations (Scopus)

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
Pages (from-to)	182-190
Number of pages	9
Journal	Biochip Journal
Volume	13
Issue number	2
DOIs	https://doi.org/10.1007/s13206-018-3209-9
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

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10.1007/s13206-018-3209-9

Cite this

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title = "Development of Hydrogel Microparticle based RT-qPCR for Advanced Detection of BCR-ABL1 Transcripts",

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).",

keywords = "BCR-ABL, Chronic myeloid leukemia, Hydrogel microparticle, N Real-time PCR, RT-qPCR, TaqMan probe",

author = "Kim, {Jung Min} and Kim, {Won Jin} and Kim, {Mi Yeon} and Kim, {Kwang Pyo} and Sim, {Sang Jun} and Kim, {Sang Kyung}",

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: {\textcopyright} 2018, The Korean BioChip Society and Springer.",

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AU - Sim, Sang Jun

AU - Kim, Sang Kyung

N1 - 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.

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N2 - 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).

AB - 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).

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Development of Hydrogel Microparticle based RT-qPCR for Advanced Detection of BCR-ABL1 Transcripts

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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