Obtaining reliable information from minute amounts of RNA using cDNA microarrays
Obtaining reliable information from minute amounts of RNA using cDNA microarrays
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DOI：
10.1186/1471-2164-3-16
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发表时间：
2002-01-01
期刊：
影响因子：
4.4
通讯作者：
Zhang, W
中科院分区：
文献类型：
--
作者：
Hu, LM;Wang, J;Zhang, W
背景：高密度 cDNA 微阵列技术提供了一种强大的工具来调查正常和患病细胞中数千个基因的活性，这有助于我们了解疾病的分子基础并确定治疗干预的潜在靶标。这项技术的前景受到实验所需的大量生物材料（每个阵列超过 50 杯总 RNA）的阻碍。我们修改了扩增程序，仅需要 1 杯总 RNA。结果分析表明，大多数使用常规方案检测为表达或差异表达的基因也可以使用扩增方案检测到。此外，使用扩增方案可以清楚地检测到许多使用常规方案未检测到或弱检测到的基因。我们通过 Northern blotting、western blotting 和免疫组织化学检测进行了一系列确认研究。结果：我们的结果表明，扩增方案揭示的大多数新信息代表了细胞中真实的基因活性。结论：我们已经证实了一种强大且一致的 cDNA 微阵列程序，可用于研究微量生物组织。
Background: High density cDNA microarray technology provides a powerful tool to survey the activity of thousands of genes in normal and diseased cells, which helps us both to understand the molecular basis of the disease and to identify potential targets for therapeutic intervention. The promise of this technology has been hampered by the large amount of biological material required for the experiments (more than 50 mug of total RNA per array). We have modified an amplification procedure that requires only 1 mug of total RNA. Analyses of the results showed that most genes that were detected as expressed or differentially expressed using the regular protocol were also detected using the amplification protocol. In addition, many genes that were undetected or weakly detected using the regular protocol were clearly detected using the amplification protocol. We have carried out a series of confirmation studies by northern blotting, western blotting, and immunohistochemistry assays.Results: Our results showed that most of the new information revealed by the amplification protocol represents real gene activity in the cells.Conclusion: We have confirmed a powerful and consistent cDNA microarray procedure that can be used to study minute amounts of biological tissue.