Reproducibility of oligonucleotide microarray transcriptome analyses -: An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae
Reproducibility of oligonucleotide microarray transcriptome analyses -: An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae
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DOI：
10.1074/jbc.m204490200
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发表时间：
2002-10-04
影响因子：
4.8
通讯作者：
Pronk, JT
中科院分区：
文献类型：
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作者：
Piper, MDW;Daran-Lapujade, P;Pronk, JT
通过使用不同的微生物菌株、培养技术和分析程序，从已发表的数据集评估DNA微阵列分析的再现性是复杂的。由于实验室内和实验室间的重现性与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，因此我们设计了一组实验来专门解决这个问题。将酿酒酵母CEN.PK113- 7 D在限定的条件下在葡萄糖限制的恒化器中生长，然后用Affyphase基因芯片阵列进行转录组分析。在每个实验室中，三个独立的重复培养物在有氧和厌氧条件下生长。尽管体外处理步骤引入的变异较小且无偏倚，但重复培养物的较大变异强调，为了获得可靠信息，实验重复至关重要。在有氧条件下，86%的最高表达的酵母基因显示出0.23的平均实验室内变异系数。这是显着低于先前报道的摇瓶培养转录组分析，可能反映了严格控制生长条件的恒化器。使用一式三份的数据集和适当的统计分析，厌氧与需氧比较的变化调用在实验室之间产生了超过95%的一致性，转录物变化超过2倍，仅留下一小部分表现出实验室偏倚的基因。
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 Gene-Chip 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 intra-laboratory 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.