Accuracy of cDNA microarray methods to detect small gene expression changes induced by neuregulin on breast epithelial cells.
Accuracy of cDNA microarray methods to detect small gene expression changes induced by neuregulin on breast epithelial cells.
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DOI：
10.1186/1471-2105-5-99
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发表时间：
2004-07-23
影响因子：
3
通讯作者：
Loeb JA
中科院分区：
文献类型：
--
作者：
Yao B;Rakhade SN;Li Q;Ahmed S;Krauss R;Draghici S;Loeb JA
cDNA微阵列是筛选生物学相关基因表达变化的有力手段，但由于随机和系统误差的“噪声”，它们准确检测微小变化的能力往往受到限制。虽然已经提出了实验设计和统计分析方法来减少这些错误，但很少有研究测试了它们的准确性和识别微小但生物学上重要的变化的能力。在这里，我们比较了两种cDNA微阵列实验设计方法和northern blot确认，以揭示基因表达的变化可能有助于神经调节蛋白对MCF10AT人乳腺上皮细胞的早期抗增殖作用。我们在相同的样品上进行了平行实验，使用带有方差分析的染料交换设计和通过逐点“校正”实验/对照杂交比率的对照/对照杂交来排除系统偏差的实验设计。我们把这种方法称为“控制校正法”（CCM）。使用重复阵列，我们发现增殖基因减少，分化基因增加。采用任意截断1.7倍且p值<0.05，我们共鉴定出32个差异表达基因，其中染料交换法9个，CCM法18个，两种方法同时使用的基因5个。这32个基因中的23个随后通过northern blotting进行了验证。其中大多数变化<2倍。虽然染料交换法（使用方差分析或贝叶斯分析）检测到的基因数量（14-16）比CCM（46）少，但它更准确（89-92%对75%）。与northern blot结果相比，对于大多数基因，微阵列结果低估了折叠变化，这意味着检测这些小变化的重要性。我们验证了cDNA微阵列实验的两种实验设计范式，这些实验能够以极好的保真度检测基因表达的微小（<2倍）变化，揭示与神经调节蛋白对MCF10AT乳腺上皮细胞的抗增殖作用相关的潜在重要基因。
cDNA microarrays are a powerful means to screen for biologically relevant gene expression changes, but are often limited by their ability to detect small changes accurately due to "noise" from random and systematic errors. While experimental designs and statistical analysis methods have been proposed to reduce these errors, few studies have tested their accuracy and ability to identify small, but biologically important, changes. Here, we have compared two cDNA microarray experimental design methods with northern blot confirmation to reveal changes in gene expression that could contribute to the early antiproliferative effects of neuregulin on MCF10AT human breast epithelial cells. We performed parallel experiments on identical samples using a dye-swap design with ANOVA and an experimental design that excludes systematic biases by "correcting" experimental/control hybridization ratios with control/control hybridizations on a spot-by-spot basis. We refer to this approach as the "control correction method" (CCM). Using replicate arrays, we identified a decrease in proliferation genes and an increase in differentiation genes. Using an arbitrary cut-off of 1.7-fold and p values <0.05, we identified a total of 32 differentially expressed genes, 9 with the dye-swap method, 18 with the CCM, and 5 genes with both methods. 23 of these 32 genes were subsequently verified by northern blotting. Most of these were <2-fold changes. While the dye-swap method (using either ANOVA or Bayesian analysis) detected a smaller number of genes (14–16) compared to the CCM (46), it was more accurate (89–92% vs. 75%). Compared to the northern blot results, for most genes, the microarray results underestimated the fold change, implicating the importance of detecting these small changes. We validated two experimental design paradigms for cDNA microarray experiments capable of detecting small (<2-fold) changes in gene expression with excellent fidelity that revealed potentially important genes associated with the anti-proliferative effects of neuregulin on MCF10AT breast epithelial cells.
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DOI：
10.1073/pnas.091062498
发表时间：
2001-04-24
影响因子：
11.1
作者：
Tusher, VG;Tibshirani, R;Chu, G
通讯作者：
Chu, G
影响因子：
1.7
作者：
Newton, MA;Kendziorski, CM;Tsui, KW
通讯作者：
Tsui, KW
影响因子：
5.8
作者：
Baldi, P;Long, AD
通讯作者：
Long, AD
影响因子：
3
作者：
Townsend, JP
通讯作者：
Townsend, JP
影响因子：
5.8
作者：
Chen, YD;Kamat, V;Trent, JM
通讯作者：
Trent, JM