Measurement of precursor miRNA in exosomes from human ESC-derived mesenchymal stem cells

Methods Mol Biol. 2013;1024:69-86. doi: 10.1007/978-1-62703-453-1_6.

Abstract

Mesenchymal stem cells (MSCs) derived from human embryonic stem cells (ESCs) have been shown to secrete exosomes that are cardioprotective against myocardial ischemia reperfusion injury in a mouse model. To elucidate this cardioprotective mechanism, we have characterized the protein, nucleic acid, and lipid composition of MSC exosomes. Here we describe the isolation and analysis of RNA in MSC exosome. We have previously reported that RNAs in MSC exosome are primarily small RNA molecules of <300 nt and they include many miRNAs. Many of these miRNAs are in the precursor form suggesting that pre-miRNAs, and not mature miRNAs are preferentially loaded into exosomes. The protocols described here include assays to ascertain the presence of pre-miRNAs, profiling of miRNA and pre-miRNA, and quantitative estimation of mature and pre-miRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation
  • Cells, Cultured
  • Culture Media, Conditioned / chemistry
  • Electrophoresis, Agar Gel
  • Embryonic Stem Cells / chemistry*
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / metabolism
  • Escherichia coli Proteins / metabolism
  • Exosomes / chemistry*
  • Exosomes / genetics
  • Humans
  • Mesenchymal Stem Cells / chemistry*
  • Mesenchymal Stem Cells / cytology
  • Mesenchymal Stem Cells / metabolism
  • MicroRNAs / isolation & purification*
  • Oligonucleotide Array Sequence Analysis
  • RNA Precursors / isolation & purification*
  • RNA Transport
  • Real-Time Polymerase Chain Reaction
  • Ribonuclease III / metabolism
  • Ultracentrifugation

Substances

  • Culture Media, Conditioned
  • Escherichia coli Proteins
  • MicroRNAs
  • RNA Precursors
  • Ribonuclease III
  • ribonuclease III, E coli