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. 2004 Feb 3;101(5):1135-40.
doi: 10.1073/pnas.0307557100. Epub 2004 Jan 22.

Normalization and analysis of cDNA microarrays using within-array replications applied to neuroblastoma cell response to a cytokine

Jianqing Fan  1 , Paul Tam, George Vande Woude, Yi Ren
Affiliations

Normalization and analysis of cDNA microarrays using within-array replications applied to neuroblastoma cell response to a cytokine

Jianqing Fan et al. Proc Natl Acad Sci U S A. .

Abstract

The quantitative comparison of two or more microarrays can reveal, for example, the distinct patterns of gene expression that define different cellular phenotypes or the genes that are induced in the cellular response to certain stimulations. Normalization of the measured intensities is a prerequisite of such comparisons. However, a fundamental problem in cDNA microarray analysis is the lack of a common standard to compare the expression levels of different samples. Several normalization protocols have been proposed to overcome the variabilities inherent in this technology. We have developed a normalization procedure based on within-array replications via a semilinear in-slide model, which adjusts objectively experimental variations without making critical biological assumptions. The significant analysis of gene expressions is based on a weighted t statistic, which accounts for the heteroscedasticity of the observed log ratios of expressions, and a balanced sign permutation test. We illustrated the use of the techniques in a comparison of the expression profiles of neuroblastoma cells that were stimulated with a growth factor, macrophage migration inhibitory factor (MIF). The analysis of expression changes at mRNA levels showed that approximately 99 genes were up-regulated and 24 were reduced significantly (P <0.001) in MIF-stimulated neuroblastoma cells. The regulated genes included several oncogenes, growth-related genes, tumor metastatic genes, and immuno-related genes. The findings provide clues as to the molecular mechanisms of MIF-mediated tumor progression and supply therapeutic targets for neuroblastoma treatment.

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Figures

Fig. 6.
Fig. 6.
(A) Estimated SD curves formula image in model 3, as a function of log intensities for six experiments. (B) The average of predicted SDs versus the SE for each gene. The correlation is ≈0.51.
Fig. 1.
Fig. 1.
Log ratios (A) and intensities (B) for pairs of clones replicated in an array connected by lines (the horizontal axis is the index of pairs). These provide useful information for normalization without making critical biological assumptions. The x axes are the indices of the pairs.
Fig. 2.
Fig. 2.
CVs computed based on intensities (A) and ratios (B), which are a measure of repeatability. The median CVs in A is 0.12 (thick bar), which is much lower than that in B (0.75). Genes with CVs above thin bar in A were filtered. (A) The average intensities against the CV based on intensities. (B) The average ratios against the CV based on ratios. (C) The average intensities versus the CV based on ratios.
Fig. 3.
Fig. 3.
(A) The intensity effects f estimated based on the data in Fig. 1 (cyan curve) and estimated by the method in ref. (green curve). (B) The unnormalized (blue) and normalized (pink) ratios for the genes with replications vs. their intensities. (C) Normalized log ratios (vs. their intensities) with intensity and print-tip effect removed for a given array, along with the lowess estimate of SDs (blue curves); two other curves are reproduced from A.
Fig. 4.
Fig. 4.
Quantile–quantile plot for log ratios Âg (A) and standardized log ratios. The standardization is effective as the quantile–quantile plot appears more linear. The x axes are the quantiles of the standard normal distribution, and y axes are the quantiles of log ratios.
Fig. 5.
Fig. 5.
The gene effect is studied by using the ordinary t test (A) and the weighted t test (B). The test statistics are plotted against their average log ratios. Genes with P values <0.001 are marked with magenta (up-regulated) and green (down-regulated). (C) Average log intensity versus log2(weighted t stat/ordinary t stat) for the genes with P values <0.001 by one of the t tests. Magenta, cyan, and green spots are the genes identified by both methods, ordinary t test only, and weighted t test, respectively. (D) The average of log intensities with MIF treatment versus those without MIF treatment. Up-regulated and down-regulated genes, identified by the weighted t test, are indicated by magenta and green, respectively.
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