Jung, Seungwon Kim, Won Jin Kim, Bong Kyun Kim, Junsun Kim, Mi Jung Kim, Kwang Pyo Kim, Sang Kyung 2024-01-19T16:33:49Z 2024-01-19T16:33:49Z 2021-09-02 2020-09-01 0956-5663 https://pubs.kist.re.kr/handle/201004/118142 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. English ELSEVIER ADVANCED TECHNOLOGY SMALL RNAS EXPRESSION PCR QUANTIFICATION In-particle stem-loop RT-qPCR for specific and multiplex microRNA profiling Article 10.1016/j.bios.2020.112301 1 BIOSENSORS & BIOELECTRONICS, v.163 BIOSENSORS & BIOELECTRONICS 163 scie scopus 000541155200020 2-s2.0-85084801899 Biophysics Biotechnology & Applied Microbiology Chemistry, Analytical Electrochemistry Nanoscience & Nanotechnology Biophysics Biotechnology & Applied Microbiology Chemistry Electrochemistry Science & Technology - Other Topics Article SMALL RNAS EXPRESSION PCR QUANTIFICATION microRNA Quantitative reverse-transcription PCR Stem-loop reverse transcription Hydrogel Multiplex assay