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| Type |
Oral Presentation |
| Area |
Oral Presentation of Young Analytical Chemists Ⅰ |
| Room No. |
303호 |
| Time |
THU 09:10-: |
| Code |
ANAL1.O-2 |
| Subject |
Hydrogel Microparticle for Highly Selective and Sensitive RT-qPCR |
| Authors |
김원진, 김상경1,*
한국과학기술연구원(KIST) 바이오마이크로시스템, Korea
1한국과학기술연구원 바이오마이크로시스템연구단, Korea
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| Abstract |
Reverse transcription – polymerase chain reaction (RT-qPCR) is commonly 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. Previously, we developed primer-immobilized network (PIN) - based RT-qPCR. PIN-based RT qPCR can capture target RNA with immobilized primers. Moreover, non-target RNA can be washed out by washing process. However, still false-positive signal was occasionally appeared in negative cases so that it can make wrong decision in clinical cases. To overcome this limitation, we improved washing protocol borrowed from a method conventionally used in a microarray. Furthermore, we established TaqMan probe-based assay instead of SYBR Green Ⅰ. We confirm that cycle threshold value (Ct value) was clearly decreased from 32 to 39~40 in negative cases. That means most non-target RNAs and interfering substances were washed away. Also, TaqMan probe-based qPCR can detect single-copy of template in a PIN particle with more than 90% of PCR efficiency. Owing to these process, PIN-based RT-qPCR can detect rare target in tens of pico grams of total RNAs. This advanced PIN RT-qPCR will soon be validated with rare target gene in clinical samples.
References
[1] Perego, R., Costantini, M., Cornacchini, M., Gargantini, L., Bianchi, C., Pungolino, E., Rovida, E., Morra, E., Eur. J. Cancer. 36, 1395–1401 (2000)
[2] Oh, E. H., Jung. S., Kim, W. J., Kim, K. P., Kim, S. K., Biosens. Bioelectron. 87, 229–235 (2017).
Keywords: Hydrogel microparticle, Rare mutation, Reverse transcription, Real-time polymerase chain reaction
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| E-mail |
wjkim@kist.re.kr |
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119th General Meeting of the KCS