Galán Amparo, Simón Carlos
Stem Cell Bank, Prince Felipe Research Center (CIPF), Valencia, Spain.
Methods Mol Biol. 2010;584:135-50. doi: 10.1007/978-1-60761-369-5_8.
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.
人类胚胎干细胞（hESC）培养需要长期维持未分化状态。实时定量聚合酶链反应（RT-PCR）技术可对目标基因进行相对定量分析，包括未分化和分化标志物，通过加入作为内部对照的校准物来比较不同批次反应，该校准物可在不同时间用于参照管家基因。主要内容包括分析所需的最少细胞数量、要检测的基因，以及如何选择合适的校准物样本和参照基因。在本章中，我们介绍如何应用RT-PCR技术、其操作流程、实验设置和软件分析，用于长期培养监测的hESC系连续传代中的基因表达分析。
