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Biosens Bioelectron. 2018 Mar 15;101:235-244. doi: 10.1016/j.bios.2017.10.039. Epub 2017 Oct 19.

Hydrogel micropost-based qPCR for multiplex detection of miRNAs associated with Alzheimer's disease.

Author information

1
Department of Chemical and Biological Engineering, Korea University, Seoul 02841, Republic of Korea; Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea.
2
Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea.
3
Department of Environmental and Safety Engineering, Ajou University, Suwon 16499, Republic of Korea.
4
Department of Chemical Engineering, Ajou University, Suwon 16499, Republic of Korea; Department of Energy System Research, Ajou University, Suwon 16499, Republic of Korea.
5
Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea; Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology (UST), Seoul 02792, Republic of Korea.
6
Department of Chemical and Biological Engineering, Korea University, Seoul 02841, Republic of Korea. Electronic address: jungkyu_choi@korea.ac.kr.
7
Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea; Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology (UST), Seoul 02792, Republic of Korea. Electronic address: nakwon.choi@kist.re.kr.

Abstract

Quantitative polymerase chain reaction (qPCR) renders profiling of genes of interest less time-consuming and cost-effective. Recently, multiplex profiling of miRNAs has enabled identifying or investigating predominant miRNAs for various diseases such as cancers and neurodegenerative diseases. Conventional multiplex qPCR technologies mostly use colorimetric measurements in solution phase, yet not only suffer from limited multiplexing capacity but also require target-screening processes due to non-specific binding between targets and primers. Here, we present hydrogel micropost-based qPCR for multiplex detection of miRNAs associated with Alzheimer's disease (AD). Our methodology promises two key advantages compared with the conventional solution-based PCR: 1) nearly no non-specific crosstalks between targets and primers, and 2) practically valuable multiplexing by spatial encoding within a single microchamber. Specifically, we immobilized hydrogel microposts (~ 400┬Ám in diameter) within commercially available polycarbonate PCR chips by multi-step ultraviolet (UV, 365nm) exposure. We optimized this photoimmobilization for thermal cycles of PCR as well. Acrylated forward primers incorporated in polyethylene glycol diacrylate (PEGDA) posts played a crucial role to confine fluorescent signal of cDNA amplification within the PEGDA hydrogel. To demonstrate the potential of our platform, we successfully verified multiplex detection of five miRNAs, which were reported to be highly correlated with AD, from a complex buffer of human plasma.

KEYWORDS:

Alzheimer's disease; Hydrogel micropost; Multiplex qPCR; Plastic substrate; Polyethylene glycol diacrylate (PEGDA); miRNA

PMID:
29096361
DOI:
10.1016/j.bios.2017.10.039
[Indexed for MEDLINE]

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