Human Embryonic Stem Cell Protocols

Volume 584 of the series Methods in Molecular Biology pp 135-150


Monitoring Stemness in Long-Term hESC Cultures by Real-Time PCR

  • Amparo GalánAffiliated withStem Cell Bank, Prince Felipe Research Center (CIPF)
  • , Carlos SimónAffiliated withStem Cell Bank, Prince Felipe Research Center (CIPF)IVI Foundation, Instituto Universitario IVI, University of Valencia

* Final gross prices may vary according to local VAT.

Get Access


Human embryonic stem cells (hESC) involve long-term cultures that must remain undifferentiated. The real-time PCR (RT-PCR) technique allows the relative quantification of target genes, including undifferentiation and differentiation markers when referred to a housekeeping control with the addition of a calibrator that serves as an internal control to compare different lots of reactions during the time. The main aspects will include a minimal number of cells to be analyzed, genes to be tested, and how to choose the appropriate calibrator sample and the reference gene. In this chapter, we present how to apply the RT-PCR technique, protocols for its performance, experimental set-up and software analysis, as of the gene expression of hESC lines in consecutive passages for long-term culture surveillance.

Key words

Real-time PCR relative quantification hESC monitoring stemnness