Molecular quantification of cell cycle-related gene expression at the protein level.
Molecular quantification of cell cycle-related gene expression at the protein level.
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DOI：
10.1002/(sici)1097-0320(20000101)39:1
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发表时间：
2000
期刊：
影响因子：
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通讯作者：
P. S. Frisa;R. Lanford;J. Jacobberger
中科院分区：
文献类型：
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作者：
P. S. Frisa;R. Lanford;J. Jacobberger
抗原和DNA含量的免疫荧光细胞计量术提供了特定表位的细胞周期时相分布的相对测量。测量信号和调节蛋白上相关表位的表达将有助于研究参与细胞周期调节和癌变的复杂途径。然而，制定调控途径模型，每个细胞的分子的测量将比强度的相对测量更有用。在这里，我们报告了一个系统，其中分子和荧光之间的关系被确定为一组参考细胞系，然后用于直接计算未知分子的数量。为了证明该过程，我们计算了参考细胞中SV 40大T抗原（Tag）的细胞周期时相分布。方法分离一组表达不同水平Tag的细胞系克隆。对这些细胞和纯化的重组Tag进行定量Western印迹。对来自同一样本的细胞进行染色并通过流式细胞术分析标签和DNA。建立了分子与荧光之间的关系，并计算了Tag的相位分布。结果通过Western印迹法测定，5种细胞系每个细胞含有0.11、0.27、1.06、2.44和2.63 × 10（6）分子的Tag。平均变异系数为10.6%。在0.11 - 2.63 x 10（6）分子范围内，分子与荧光的关系符合线性方程（r（2）= 0.96），然而，相同的方程不符合0分子（由同种型染色对照定义）与最低表达细胞系之间的关系。为了计算最低细胞系中分子的相分布，使用从0到110，000个分子的第二线性方程。结论：这项工作描述了一个系统，其中固定的细胞表达不同水平的目标抗原定量Western印迹可用于标准化流式细胞术测量的基因表达的绝对值。
BACKGROUND Immunofluorescence cytometry of antigen and DNA content provides relative measurements of the cell cycle phase distribution of a specific epitope. Measurement of correlated expression of epitopes on signaling and regulatory proteins will be useful in the study of the complex pathways involved in cell cycle regulation and carcinogenesis. However, to formulate regulatory pathway models, measurements of molecules per cell would be more useful than relative measurements of intensity. Here, we report on a system in which the relationship between molecules and fluorescence is determined for a reference set of cell lines that are then used to directly calculate the number of molecules for unknowns. To demonstrate the process, we calculated the cell cycle phase distribution of SV40 large T antigen (Tag) in the reference cells. METHODS A set of cell line clones expressing different levels of Tag were isolated. Quantitative Western blots of these cells and purified, recombinant Tag were performed. Cells from the same sample were stained and analyzed by flow cytometry for Tag and DNA. The relationship between molecules and fluorescence was established and calculations were performed for the phase distributions of Tag. RESULTS The five cell lines had 0.11, 0.27, 1.06, 2.44, and 2.63 x 10(6) molecules of Tag per cell, determined by Western blot. The average coefficient of variation was 10.6%. The relationship of molecules to fluorescence fit a linear equation (r(2) = 0.96) over the range, 0.11 - 2.63 x 10(6) molecules, however, the same equation did not fit the relationship between 0 molecules, defined by isotype staining controls, and the lowest expressing cell line. To calculate the phase distributions of molecules in the lowest cell line, a second linear equation from 0 to 110,000 molecules was used. CONCLUSIONS This work describes a system where fixed cells expressing various levels of a target antigen quantified by Western blots can be used to standardize flow cytometric measurements of gene expression in absolute terms.