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

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 react...

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Bibliographic Details
Published inBiochip journal Vol. 13; no. 2; pp. 182 - 190
Main Authors Kim, Jung Min, Kim, Won Jin, Kim, Mi Yeon, Kim, Kwang Pyo, Sim, Sang Jun, Kim, Sang Kyung
Format Journal Article
LanguageEnglish
Published Seoul The Korean Society for Applied Biological Chemistry 01.06.2019
Springer Nature B.V
한국바이오칩학회
Subjects
Binding sites
Biomedical Engineering and Bioengineering
Biotechnology
Cancer
Chemistry
Design
Efficiency
Genes
Hybridization
Hydrogels
Kinases
Microparticles
Mutation
Original Article
Polymerase chain reaction
Reverse transcription
Ribonucleic acid
RNA
Target detection
생물공학
United States > US
TaqMan probe
N Real-time PCR
Chronic myeloid leukemia
Hydrogel microparticle
RT-qPCR
BCR-ABL
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Summary: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).
ISSN:1976-0280
2092-7843
DOI:10.1007/s13206-018-3209-9