Piper Matthew D W, Daran-Lapujade Pascale, Bro Christoffer, Regenberg Birgitte, Knudsen Steen, Nielsen Jens, Pronk Jack T
Kluyver Laboratory of Biotechnology, Technical University of Delft, Julianalaan 26, Delft 2628BC, The Netherlands.
J Biol Chem. 2002 Oct 4;277(40):37001-8. doi: 10.1074/jbc.M204490200. Epub 2002 Jul 16.
Assessment of reproducibility of DNA-microarray analysis from published data sets is complicated by the use of different microbial strains, cultivation techniques, and analytical procedures. Because intra- and interlaboratory reproducibility is highly relevant for application of DNA-microarray analysis in functional genomics and metabolic engineering, we designed a set of experiments to specifically address this issue. Saccharomyces cerevisiae CEN.PK113-7D was grown under defined conditions in glucose-limited chemostats, followed by transcriptome analysis with Affymetrix GeneChip arrays. In each of the laboratories, three independent replicate cultures were grown aerobically as well as anaerobically. Although variations introduced by in vitro handling steps were small and unbiased, greater variation from replicate cultures underscored that, to obtain reliable information, experimental replication is essential. Under aerobic conditions, 86% of the most highly expressed yeast genes showed an average intralaboratory coefficient of variation of 0.23. This is significantly lower than previously reported for shake-flask-culture transcriptome analyses and probably reflects the strict control of growth conditions in chemostats. Using the triplicate data sets and appropriate statistical analysis, the change calls from anaerobic versus aerobic comparisons yielded an over 95% agreement between the laboratories for transcripts that changed by over 2-fold, leaving only a small fraction of genes that exhibited laboratory bias.
已发表数据集中DNA微阵列分析的可重复性评估因使用不同的微生物菌株、培养技术和分析程序而变得复杂。由于实验室内部和实验室间的可重复性对于DNA微阵列分析在功能基因组学和代谢工程中的应用高度相关,我们设计了一组实验来专门解决这个问题。酿酒酵母CEN.PK113 - 7D在限定条件下于葡萄糖限制的恒化器中培养,随后用Affymetrix基因芯片阵列进行转录组分析。在每个实验室中,进行了三个独立的重复培养,包括好氧培养和厌氧培养。尽管体外操作步骤引入的变异很小且无偏差,但重复培养之间的变异更大,这突出表明,为了获得可靠的信息,实验重复是必不可少的。在好氧条件下,86%表达量最高的酵母基因显示实验室内部平均变异系数为0.23。这显著低于先前报道的摇瓶培养转录组分析结果,可能反映了恒化器中生长条件的严格控制。使用三份数据集并进行适当的统计分析,厌氧与好氧比较得出的变化调用在实验室之间对于变化超过2倍的转录本有超过95%的一致性,只有一小部分基因表现出实验室偏差。