Dumur Catherine I, Nasim Suhail, Best Al M, Archer Kellie J, Ladd Amy C, Mas Valeria R, Wilkinson David S, Garrett Carleton T, Ferreira-Gonzalez Andrea
Department of Pathology, Virginia Commonwealth University, Richmond, VA 23298, USA.
Clin Chem. 2004 Nov;50(11):1994-2002. doi: 10.1373/clinchem.2004.033225. Epub 2004 Sep 13.
Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy.
In the present studies we developed quality-control criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A(260)/A(280) ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation.
Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01).
These results confirm the need to establish defined quality-control criteria for sample quality to distinguish between analytical and biological variability.
为确保微阵列结果在潜在临床应用中的可重复性而制定质量控制标准仍处于起步阶段。
在本研究中，我们制定了质量控制标准，并使用来自细胞系、冷冻肿瘤和市售参考RNA的总RNA评估了它们在微阵列数据分析中的效果。评估了诸如A(260)/A(280)比值、rRNA百分比以及cDNA和cRNA合成产物的中位大小等质量控制标准在微阵列分析中的稳健性。此外，还在Affymetrix HG-U133A高密度寡核苷酸微阵列上使用参考材料进行了精密度研究。在Affymetrix流体工作站的四个不同模块中，在两天内运行的16个不同芯片上检测了相同的参考RNA样本。还比较了新鲜和冷冻的片段化cRNA。拟合了一个方差分析模型以确定主要变异来源。
高质量样本在电泳图中显示rRNA>30%，cDNA和cRNA合成产物的中位大小分别为2.0和3.0 kb。精密度研究表明，主要变异来源是每日变异性，对杂交外源对照基因的影响最小。总体而言，结果表明当使用符合质量控制标准的RNA时，Affymetrix基因芯片系统具有高度可重复性（总体P>0.01）。
这些结果证实了需要为样本质量建立明确的质量控制标准，以区分分析变异性和生物学变异性。
