DISTRIBUTION AND CLONING OF EUKARYOTIC MESSENGER-RNAS BY MEANS OF DIFFERENTIAL DISPLAY - REFINEMENTS AND OPTIMIZATION
DISTRIBUTION AND CLONING OF EUKARYOTIC MESSENGER-RNAS BY MEANS OF DIFFERENTIAL DISPLAY - REFINEMENTS AND OPTIMIZATION
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DOI：
10.1093/nar/21.14.3269
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发表时间：
1993-07-11
影响因子：
14.9
通讯作者：
PARDEE, AB
中科院分区：
文献类型：
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作者：
LIANG, P;AVERBOUKH, L;PARDEE, AB
差异显示已被开发为检测和表征真核细胞中改变的基因表达的工具。基本原理是系统地扩增信使 RNA，然后将其 3' 末端分布在变性聚丙烯酰胺凝胶上。在这里，我们提供方法学细节，并深入检查该方法的特异性、敏感性和重现性。我们表明，锚定的oligo-dT引物的数量可以从12个减少到4个，这些引物在3'端倒数第二个碱基处简并。我们还证明，使用此处描述的优化条件，可以同时显示来自相关细胞的多个 RNA 样本。因此，可以更准确地识别过程特异性基因而不是细胞特异性基因。这些结果能够进一步简化该技术，并使其易于适用于广泛的生物系统。
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.