Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples
Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples
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DOI：
10.1093/hmg/8.9.1715
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发表时间：
1999-09-01
影响因子：
3.5
通讯作者：
Jordan, BR
中科院分区：
文献类型：
--
作者：
Bertucci, F;Bernard, K;Jordan, BR
DNA 或寡核苷酸阵列广泛用于大规模表达测量，使用各种实施方式：一方面，将 DNA 点样到相对较大尺寸的尼龙膜上（具有放射性检测）；另一方面，载玻片上的微阵列和寡核苷酸芯片都与荧光探针一起使用。最近还描述了具有比色检测功能的尼龙微阵列。与宏阵列相比，小型化系统的物理尺寸较小，允许较小的杂交体积（2-100μl）并提供较高的探针浓度。然而，我们表明，所有这些系统的实际灵敏度（定义为检测给定 mRNA 种类所需的样品量）实际上是相似的，这主要是由于各个阵列上存在的目标材料量差异很大。然后，我们证明尼龙微阵列与 P-33 标记的放射性探针的组合提供了 100 倍的灵敏度，使得使用来自小生物样品的亚微克量的未扩增总 RNA 进行表达谱实验成为可能。这对基础和临床研究具有重要意义，并使这种替代方法特别适合在学术背景下运作的团体。
DNA or oligonucleotide arrays are widely used for large-scale expression measurements, using various implementations: macroarrays in which DNA is spotted onto nylon membranes of relatively large dimensions (with radioactive detection) on the one hand; microarrays on glass slides and oligonucleotide chips, both used with fluorescent probes, on the other hand. Nylon microarrays with colourimetric detection have also been described recently. The small physical dimensions of miniaturized systems allow small hybridization volumes (2-100 mu l) and provide high probe concentrations, in contrast to macroarrays. We show, however, that actual sensitivity (defined as the amount of sample necessary for detection of a given mRNA species) is in fact similar for ail these systems and that this is mostly due to the very different amounts of target material present on the respective arrays. We then demonstrate that the combination of nylon microarrays with P-33-labelled radioactive probes provides 100-fold better sensitivity, making it possible to perform expression profiling experiments using submicrogram amounts of unamplified total RNA from small biological samples. This has important implications in basic and clinical research and makes this alternative approach particularly suitable for groups operating in an academic context.