Molecular beacon-enabled purification of living cells by targeting cell type-specific mRNAs

Nat Protoc. 2014 Oct;9(10):2411-24. doi: 10.1038/nprot.2014.154. Epub 2014 Sep 18.


Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics, the MB-based method enables the isolation of a wide variety of cells. For example, the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design, validation and nucleofection into cells, we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol, we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with ∼ 97% purity, as confirmed by electrophysiology and immunocytochemistry. After designing MBs, their ordering and validation requires 2 weeks, and the isolation process requires 3 h.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • Cell Differentiation
  • Cell Separation / instrumentation
  • Cell Separation / methods*
  • Cells, Cultured
  • Electrophysiology / methods
  • Flow Cytometry / methods
  • Humans
  • Mice
  • Molecular Biology / instrumentation
  • Molecular Biology / methods*
  • Molecular Probes / chemistry
  • Myocytes, Cardiac / cytology
  • Oligonucleotides / chemistry
  • Oligonucleotides / metabolism*
  • Organ Specificity
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / physiology
  • RNA, Messenger / analysis
  • RNA, Messenger / metabolism*


  • Biomarkers
  • Molecular Probes
  • Oligonucleotides
  • RNA, Messenger