In-particle Stem-loop RT-qPCR for Specific and Multiplexed MicroRNA Profiling

Title
In-particle Stem-loop RT-qPCR for Specific and Multiplexed MicroRNA Profiling
Authors
김상경김준선김봉균정승원김원진Mi Jung KimKwang Pyo Kim
Keywords
microRNA; Quantitative reverse-transcription PCR; Stem-loop reverse transcription; Hydrogel; Multiplex assay
Issue Date
2020-09
Publisher
Biosensors and bioelectronics
Citation
VOL 163-7
Abstract
Here we report a novel method of microRNA (miRNA) profiling with particle-based multiplex quantitative reverse transcription polymerase chain reaction (RT-qPCR). To achieve target-specific reaction in a particle, the stem-loop RT primer and forward primer for each target miRNA were chemically immobilized to the particle. Target-specific cDNA synthesis proceeds with the stem-loop RT primer and then qPCR subsequently proceeds with the forward primer to rapidly achieve a quantitative result. High-fidelity multiplex assay was also accomplished in a single PCR process by loading multiple particles for each specific miRNA. The method for primer supply in the particles, involving confinement of the target-specific RT and PCR primers in the matrix of particles, led to the reduction of nonspecific reactions and improved the selectivity of the miRNA assay while minimizing labor in a multiple target assay. Specifically, this particle-based assay enabled the differentiation of mature miRNA from precursor with selectivity of 270:1 in terms of amplification speed. This advanced method also showed good discrimination among highly homologous let-7 family members, with cross-reaction rates of less than 5%. We demonstrated a very simple process of five-plex miRNA profiling in total RNA, and the measured changes in expression level were consistent with those from a conventional singleplex method.
URI
https://pubs.kist.re.kr/handle/201004/71839
ISSN
0956-5663
Appears in Collections:
KIST Publication > Article
Files in This Item:
There are no files associated with this item.
Export
RIS (EndNote)
XLS (Excel)
XML


qrcode

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

BROWSE