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Embryonic stem (ES) cell lines are important for use in developmental biology studies, and because these cells are totipotent, they may provide a much-needed source of differentiated cells for certain therapeutic applications. The phenotype of the ES cell in culture is often assessed by (semi)quantitative RNA analyses. In such cases, it is critical to use appropriate internal standards to correct for experimentally induced sources of error. This is particularly true for ES cell differentiation because it is heterogeneous in nature. We describe protocols for determining the suitability of housekeeping genes to act as internal controls in differentiating ES cell cultures. Such assessment is needed for every experimental condition under investigation. The protocol focuses on polymerase chain reaction; however, the principle and experimental design are applicable to any (semi)quantitative RNA assay.

Original publication

DOI

10.1385/1-59745-037-5:101

Type

Journal article

Journal

Methods in molecular biology (Clifton, N.J.)

Publication Date

01/2006

Volume

329

Pages

101 - 112

Addresses

Kennedy Institute of Rheumatology, Imperial College, London, UK.

Keywords

Cell Line, Totipotent Stem Cells, Animals, Mice, RNA, Cell Culture Techniques, Coculture Techniques, Reverse Transcriptase Polymerase Chain Reaction, Cell Differentiation, Gene Expression, Pregnancy, Female, Embryo, Mammalian