Selection of Robust Reference Genes for Normalization of Quantitative RT-PCR Data from Differentiating Human Pluripotent Stem Cells

摘要

Human pluripotent stem cells are a unique scientific and medical resource that has gained massive interest from investigators. These cells can differentiate to all cell types in the human body and hold great promise for both research and health care. The last two decades large investments have been made to understand how the differentiation process of pluripotent stem cells is controlled and to gain more knowledge, work in progress performs extensive characterizations of transcriptional and functional properties of human pluripotent stem cells and their derivatives. One of the most frequently used techniques for monitoring pluripotency and differentiation of stem cells is quantitative real time RT-PCR (qRT-PCR), which requires an accurate normalization procedure to correct for experimental variations. Commonly, researchers use housekeeping genes as endogenous controls for calibration. However, several recent reports have shown that stem cells deviate substantially from somatic cells in the expression of housekeeping genes, which may have important consequences. This paper investigates the expression of commonly used housekeeping genes in somatic cell types and explores their expression in samples from human pluripotent stem cells. Results reveal significant differences between somatic cells and stem cells that will have important implications on the selection of reliable reference genes for normalization of data from differentiating human pluripotent stem cells.