Extensible Multiplex Real-time PCR of MicroRNA Using Microparticles

Sci Rep. 2016 Mar 11:6:22975. doi: 10.1038/srep22975.

Abstract

Multiplex quantitative real-time PCR (qPCR), which measures multiple DNAs in a given sample, has received significant attention as a mean of verifying the rapidly increasing genetic targets of interest in single phenotype. Here we suggest a readily extensible qPCR for the expression analysis of multiple microRNA (miRNA) targets using microparticles of primer-immobilized networks as discrete reactors. Individual particles, 200~500 μm in diameter, are identified by two-dimensional codes engraved into the particles and the non-fluorescent encoding allows high-fidelity acquisition of signal in real-time PCR. During the course of PCR, the amplicons accumulate in the volume of the particles with high reliability and amplification efficiency over 95%. In a quick assay comprising of tens of particles holding different primers, each particle brings the independent real-time amplification curve representing the quantitative information of each target. Limited amount of sample was analyzed simultaneously in single chamber through this highly multiplexed qPCR; 10 kinds of miRNAs from purified extracellular vesicles (EVs).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell-Derived Microparticles / chemistry
  • Cell-Derived Microparticles / genetics
  • Extracellular Vesicles / genetics
  • Gene Expression Regulation / genetics
  • Humans
  • MicroRNAs / biosynthesis
  • MicroRNAs / genetics
  • MicroRNAs / isolation & purification*
  • Multiplex Polymerase Chain Reaction / methods*
  • Real-Time Polymerase Chain Reaction / methods*

Substances

  • MicroRNAs