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

Autores de CIPF

• 

  Amparo Galán Albiñana

  Autor

• Carlos Simón Vallés

  Autor

Grupos de Investigación

• Laboratorio de Neuroendocrinología Molecular

  

  Jefe/a de Grupo

  Deborah J Burks

Abstract

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 setup, and software analysis, as of the gene expression of hESC lines in consecutive passages for long-term culture surveillance.