Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays
Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays
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DOI：
10.1093/nar/30.10.e48
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发表时间：
2002-05-15
影响因子：
14.9
通讯作者：
Sealfon, SC
中科院分区：
文献类型：
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作者：
Yuen, T;Wurmbach, E;Sealfon, SC
我们通过使用已知调节两种阵列上测量的许多基因的范式分析实验中的测试RNA样本，比较了使用寡核苷酸阵列（GeneChip，Affymetrix）获得的微阵列测量结果与实验室开发的cDNA阵列的准确性。我们选择了47个基因代表的两个阵列，包括已知的调控和非调控的转录本，并建立了参考相对表达测量这些基因在测试RNA样品中使用定量逆转录酶实时PCR（QRTPCR）测定。通过应用新的数学模型建立了可重现性（平均变异系数= 11.8%）QRTPCR测量的有效性。两种阵列平台在鉴定调控和非调控基因方面的性能是相同的。使用任一平台，17个明确调控的基因中的16个被正确鉴定，并且没有明确不调控的转录物被错误地鉴定为调控的。通过确定测量偏倚，评估每个平台获得的倍数变化测量值的准确度。两个平台一致低估了实验和对照样品之间mRNA表达的相对变化。用cDNA阵列观察到的偏倚对于倍数变化是可预测的
We compared the accuracy of microarray measurements obtained with oligonucleotide arrays (GeneChip, Affymetrix) with a laboratory-developed cDNA array by assaying test RNA samples from an experiment using a paradigm known to regulate many genes measured on both arrays. We selected 47 genes represented on both arrays, including both known regulated and unregulated transcripts, and established reference relative expression measurements for these genes in the test RNA samples using quantitative reverse transcriptase real-time PCR (QRTPCR) assays. The validity of the reproducible (average coefficient of variation = 11.8%) QRTPCR measurements were established through application of a new mathematical model. The performance of both array platforms in identifying regulated and non-regulated genes was identical. With either platform, 16 of 17 definitely regulated genes were correctly identified, and no definitely unregulated transcript was falsely identified as regulated. Accuracy of the fold-change measurements obtained with each platform was assessed by determining measurement bias. Both platforms consistently underestimate the relative changes in mRNA expression between experimental and control samples. The bias observed with cDNA arrays was predictable for fold-changes