Ma Changqing, Lyons-Weiler Maureen, Liang Wenjing, LaFramboise William, Gilbertson John R, Becich Michael J, Monzon Federico A
Department of Pathology, Center for Pathology Informatics, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15237, USA.
J Mol Diagn. 2006 May;8(2):183-92. doi: 10.2353/jmoldx.2006.050077.
The effect of different amplification and labeling methods on DNA microarray expression results has not been previously delineated. To analyze the variation associated with widely accepted T7-based RNA amplificationand labeling methods, aliquots of the Stratagene Human Universal Reference RNA were labeled using three eukaryotic target preparation methods followed by uniform replicate array hybridization (Affymetrix U95Av2). Method-dependent variability was observed in the yield and size distribution of labeled products, as well as in the gene expression results. A significant increase in short transcripts, when compared to unamplified mRNA, was observed in methods with long in vitro transcription reactions. Intramethod reproducibility showed correlation coefficients >0.99, whereas intermethod comparisons showed coefficients ranging from 0.94 to 0.98 and a nearly twofold increase in coefficient of variation. Fold amplification for each method positively correlated with the number of genes present. Our experiments uncovered two factors that introduced significant bias in gene expression data: the number of labeled nucleotides, which introduces sequence-dependent bias, and the length of the in vitro transcription reaction, which introduces transcript size-dependent bias. This study provides evidence that variability in expression data may be caused, in part, by differences in amplification and labeling protocols.
不同扩增和标记方法对DNA微阵列表达结果的影响此前尚未明确。为分析与广泛接受的基于T7的RNA扩增和标记方法相关的变异，使用三种真核生物靶标制备方法对Stratagene人类通用参考RNA的等分试样进行标记，随后进行均匀重复阵列杂交（Affymetrix U95Av2）。在标记产物的产量和大小分布以及基因表达结果中观察到了方法依赖性变异。在进行长时间体外转录反应的方法中，与未扩增的mRNA相比，短转录本显著增加。方法内重复性显示相关系数>0.99，而方法间比较显示系数范围为0.94至0.98，变异系数增加了近两倍。每种方法的扩增倍数与存在的基因数量呈正相关。我们的实验发现了两个在基因表达数据中引入显著偏差的因素：引入序列依赖性偏差的标记核苷酸数量，以及引入转录本大小依赖性偏差的体外转录反应长度。这项研究提供了证据，表明表达数据的变异性可能部分是由扩增和标记方案的差异引起的。
