Crawford Dana R, Kochheiser John C, Schools Gary P, Salmon Sharon L, Davies Kelvin J A
Center for Immunology and Microbial Disease, The Albany Medical College, NY 12208, USA.
Gene Expr. 2002;10(3):101-7.
Differential display (DD) is a well-established analytical tool for measuring gene expression that is still popular due to its documented success and ability to identify novel genes not yet available for analysis by more powerful microarray hybridization. For a comprehensive analysis of all mRNAs in a given cell, it is statistically predicted that at least 240 different DD primer combinations are required. This prediction, however, has never been empirically tested. Using far more primer combinations than that predicted to evaluate 90% of the mRNAs in a cell, plus other modifications, we identified and confirmed the induction of five mRNAs by hydrogen peroxide in HA-1 hamster cells. However, five other known oxidant-inducible mRNAs were not identified by DD. Filter microarray hybridization did not result in the identification of any additional species modulated twofold or greater but previous two-dimensional protein gel electrophoresis identified 15 induced protein species. We conclude that the current statistical prediction for comprehensive analysis of all the mRNAs in a given cell is inaccurate, at least in our hands, and further conclude that DD is a useful but less than comprehensive method for assessing changes in mRNA levels.
差异显示（DD）是一种成熟的用于测量基因表达的分析工具，由于其已被证实的成功以及能够识别出用更强大的微阵列杂交技术尚无法分析的新基因，至今仍广受欢迎。对于给定细胞中所有mRNA的全面分析，据统计预测至少需要240种不同的DD引物组合。然而，这一预测从未经过实证检验。我们使用了比预测数量多得多的引物组合来评估细胞中90%的mRNA，并进行了其他改进，从而在HA - 1仓鼠细胞中鉴定并证实了过氧化氢对五种mRNA的诱导作用。然而，差异显示法未鉴定出其他五种已知的可被氧化剂诱导的mRNA。滤膜微阵列杂交未鉴定出任何其他上调两倍或更高倍数的物种，但之前的二维蛋白质凝胶电泳鉴定出了15种诱导蛋白物种。我们得出结论，目前对于给定细胞中所有mRNA进行全面分析的统计预测是不准确的，至少在我们的实验中如此，并且进一步得出结论，差异显示法是一种有用但不够全面的评估mRNA水平变化的方法。
