Daly Thomas M, Dumaual Carmen M, Dotson Crystal A, Farmen Mark W, Kadam Sunil K, Hockett Richard D
Division of Experimental Medicine, Genomic Medicine Group, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46240, USA.
J Mol Diagn. 2005 Aug;7(3):404-12. doi: 10.1016/S1525-1578(10)60570-3.
Although gene expression profiling using microarray technology is widely used in research environments, adoption of microarray testing in clinical laboratories is currently limited. In an attempt to determine how such assays would perform in a clinical laboratory, we evaluated the analytical variability of Affymetrix microarray probesets using two generations of human Affymetrix chips (U95Av2 and U133A). The study was designed to mimic potential clinical applications by using multiple operators, machines, and reagent lots, and by performing analyses throughout a period of several months. A mixed model analysis was used to evaluate the relative contributions of multiple factors to overall variability, including operator, instrument, run, cRNA/cDNA synthesis, and changes in reagent lots. Under these conditions, the average probeset coefficient of variation (CV) was relatively low for present probesets on both generations of chips (mean coefficient of variation, 21.9% and 27.2% for U95Av2 and U133A chips, respectively). The largest contribution to overall variation was chip-to-chip (residual) variability, which was responsible for between 40 to 60% of the total variability observed. Changes in individual reagent lots and instrumentation contributed very little to the overall variability. We conclude that the approach demonstrated here could be applied to clinical validation of Affymetrix-based assays and that the analytical precision of this technique is sufficient to answer many biological questions.
尽管利用微阵列技术进行基因表达谱分析在研究环境中被广泛应用，但目前临床实验室采用微阵列检测的情况有限。为了确定此类检测在临床实验室中的表现，我们使用两代人类Affymetrix芯片（U95Av2和U133A）评估了Affymetrix微阵列探针集的分析变异性。该研究旨在通过使用多名操作人员、多台机器和多个试剂批次，并在几个月的时间内进行分析，来模拟潜在的临床应用。采用混合模型分析来评估多个因素对总体变异性的相对贡献，这些因素包括操作人员、仪器、检测批次、cRNA/cDNA合成以及试剂批次的变化。在这些条件下，两代芯片上已检测到的探针集的平均变异系数（CV）相对较低（U95Av2和U133A芯片的平均变异系数分别为21.9%和27.2%）。对总体变异贡献最大的是芯片间（残差）变异性，其占观察到的总变异性的40%至60%。单个试剂批次和仪器的变化对总体变异性的贡献非常小。我们得出结论，此处展示的方法可应用于基于Affymetrix检测的临床验证，并且该技术的分析精度足以回答许多生物学问题。
