Liang P, Averboukh L, Pardee A B
Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, MA.
Nucleic Acids Res. 1993 Jul 11;21(14):3269-75. doi: 10.1093/nar/21.14.3269.
Differential display has been developed as a tool to detect and characterize altered gene expression in eukaryotic cells. The basic principle is to systematically amplify messenger RNAs and then distribute their 3' termini on a denaturing polyacrylamide gel. Here we provide methodological details and examine in depth the specificity, sensitivity and reproducibility of the method. We show that the number of anchored oligo-dT primers can be reduced from twelve to four that are degenerate at the penultimate base from the 3' end. We also demonstrate that using optimized conditions described here, multiple RNA samples from related cells can be displayed simultaneously. Therefore process-specific rather than cell-specific genes could be more accurately identified. These results enable further streamlining of the technique and make it readily applicable to a broad spectrum of biological systems.
差异显示技术已被开发为一种用于检测和表征真核细胞中基因表达变化的工具。其基本原理是系统地扩增信使核糖核酸，然后将其3'末端分布在变性聚丙烯酰胺凝胶上。在此，我们提供了方法学细节，并深入研究了该方法的特异性、灵敏度和可重复性。我们表明，锚定寡聚dT引物的数量可以从12个减少到4个，这些引物在3'末端的倒数第二个碱基处是简并的。我们还证明，使用此处所述的优化条件，可以同时展示来自相关细胞的多个RNA样本。因此，可以更准确地鉴定过程特异性而非细胞特异性基因。这些结果使该技术能够进一步简化，并使其易于应用于广泛的生物系统。
