Automated target preparation for microarray-based gene expression analysis
Automated target preparation for microarray-based gene expression analysis
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DOI：
10.1021/ac060097t
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发表时间：
2006-09-15
影响因子：
7.4
通讯作者：
Kussmann, Martin
中科院分区：
文献类型：
--
作者：
Raymond, Frederic;Metairon, Sylviane;Kussmann, Martin
DNA 微阵列已迅速发展成为大规模并行基因表达分析的平台。尽管该技术被广泛使用，但仍被批评容易受到技术变化的影响。针对这个问题，最近的比较、跨平台和实验室间研究表明，给定“湿实验室”实验和数据处理的明确程序，可以实现令人满意的再现性和很小的实验变异性。鉴于标准化方面的这些进步，对统一样品制备的要求变得显而易见，特别是如果微阵列平台被用作设施，即由在实验室工作的不同用户使用。虽然减少技术变异性的一种选择是让一名实验室技术人员专门负责所有微阵列研究，但我们决定通过与热循环仪和微量滴定板读数器耦合的液体处理系统来实现整个 RNA 样品制备的自动化。事实上，芯片分析之前的自动化 RNA 样品制备能够（1）减少实验引起的结果变异性，（2）将（重要的）生物变异性与（不需要的）实验变异性分开，以及（3）基因表达结果的研究间比较。我们的机器人平台可以使用自动化样品制备方法并行处理多达 24 个样品，该方法可产生高质量的生物素标记 cRNA，准备在 Affymetrix GeneChips 上进行杂交。结果表明，技术实验间的差异不如手动制备的样品明显。此外，使用相同起始材料的实验表明，自动化过程在样品之间产生了良好的重现性。
DNA microarrays have rapidly evolved toward a platform for massively paralleled gene expression analysis. Despite its widespread use, the technology has been criticized to be vulnerable to technical variability. Addressing this issue, recent comparative, interplatform, and interlaboratory studies have revealed that, given defined procedures for "wet lab" experiments and data processing, a satisfactory reproducibility and little experimental variability can be achieved. In view of these advances in standardization, the requirement for uniform sample preparation becomes evident, especially if a microarray platform is used as a facility, i.e., by different users working in the laboratory. While one option to reduce technical variability is to dedicate one laboratory technician to all microarray studies, we have decided to automate the entire RNA sample preparation implementing a liquid handling system coupled to a thermocycler and a microtiter plate reader. Indeed, automated RNA sample preparation prior to chip analysis enables ( 1) the reduction of experimentally caused result variability, ( 2) the separation of ( important) biological variability from ( undesired) experimental variation, and ( 3) interstudy comparison of gene expression results. Our robotic platform can process up to 24 samples in parallel, using an automated sample preparation method that produces high-quality biotin-labeled cRNA ready to be hybridized on Affymetrix GeneChips. The results show that the technical interexperiment variation is less pronounced than with manually prepared samples. Moreover, experiments using the same starting material showed that the automated process yields a good reproducibility between samples.