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

Authors
 Brian M. Wile  ;  Kiwon Ban  ;  Young-Sup Yoon  ;  Gang Bao 
Citation
 NATURE PROTOCOLS, Vol.9(10) : 2411-2424, 2014 
Journal Title
 NATURE PROTOCOLS 
ISSN
 1754-2189 
Issue Date
2014
MeSH
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*
Abstract
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.
Files in This Item:
T201405947.pdf Download
DOI
10.1038/nprot.2014.154
Appears in Collections:
1. College of Medicine (의과대학) > BioMedical Science Institute (의생명과학부) > 1. Journal Papers
Yonsei Authors
Yoon, Young Sup(윤영섭)
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/139010
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