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

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. Conven...

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Published in:Biosensors & bioelectronics 2018-03, Vol.101, p.235-244
Main Authors: Choi, Woongsun, Yeom, Sang Yun, Kim, Junsun, Jung, Seungwon, Jung, Seungho, Shim, Tae Soup, Kim, Sang Kyung, Kang, Ji Yoon, Lee, Soo Hyun, Cho, Il-Joo, Choi, Jungkyu, Choi, Nakwon
Format: Article
Language:English
Subjects:
Alzheimer Disease - blood
Alzheimer Disease - genetics
Alzheimer's disease
Biosensing Techniques - methods
DNA, Complementary - genetics
Humans
Hydrogel micropost
Hydrogel, Polyethylene Glycol Dimethacrylate - chemistry
MicroRNAs - analysis
MicroRNAs - blood
MicroRNAs - genetics
miRNA
Multiplex Polymerase Chain Reaction - methods
Multiplex qPCR
Plastic substrate
Polyethylene glycol diacrylate (PEGDA)
Polyethylene Glycols - chemistry
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Summary: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 (~ 400µm 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. [Display omitted] •Hydrogel micropost-based multiplex qPCR is reported for detection of miRNAs.•Multi-step UV-exposure allows for immobilization of PEGDA microposts in PCR chips.•Hydrogel-based qPCR results in the significant elimination of non-specific binding.•5-plex detection of miRNAs is presented with excellent specificity & selectivity.
ISSN:0956-5663
1873-4235
DOI:10.1016/j.bios.2017.10.039