External RNA controls (ERCs), although important for microarray assay performance assessment, have yet to be fully implemented in the research community. As part of the MicroArray Quality Control (MAQC) study, two types of ERCs were implemented and evaluated; one was added to the total RNA in the samples before amplification and labeling; the other was added to the copyRNAs (cRNAs) before hybridization. ERC concentration-response curves were used across multiple commercial microarray platforms to identify problematic assays and potential sources of variation in the analytical process. In addition, the behavior of different ERC types was investigated, resulting in several important observations, such as the sample-dependent attributes of performance and the potential of using these control RNAs in a combinatorial fashion. This multiplatform investigation of the behavior and utility of ERCs provides a basis for articulating specific recommendations for their future use in evaluating assay performance across multiple platforms.
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Comparison of the correlation coefficients for each assay from the lines fit through the expected versus observed log10 ratios in Figure 4. (DOC 99 kb)
The tERC signal intensity across different RNA samples. (DOC 89 kb)
The effect of normalization methods on the tERC performance behavior across the same RNA samples illustrated in Supplementary Figure 2. (DOC 141 kb)
Observed log10 ratios for the AGL tERCs that are spiked in at intended 1:1 ratios in the Two-Color hybridization samples. (DOC 34 kb)
Correlation Assuming Percent Brain is Changed to mRNA Differences Between in the Samples. (DOC 110 kb)
The cERC signal intensity (y-axis) was compared across the four different RNA samples (A, B, C and D) for the ABI (top graph) and AFX (bottom graph) platforms. (DOC 34 kb)
The effect of normalization method on the sample independency of the cERC signal intensity (y-axis) for the AFX microarray platform. (DOC 66 kb)
Hierarchical cluster analysis for the One-Color AG1 platform based on either the tERC probes (A) or the biological probes (B). (DOC 157 kb)
Full Concentration-Response Curves for tERCs on the Agilent microarray platform. (DOC 267 kb)
Summary of cERC Concentration and tERC Molar Ratio Used for Plotting Concentration-Response Curves in Figure 1. (DOC 52 kb)
Summary of statistical results presented in Figure 3. (DOC 1945 kb)
Summary of statistical results presented in Figure 5. (DOC 39 kb)
Summary of tERC Concentration and Expected Two-Color Ratios for the AGL Platform. (DOC 39 kb)
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Tong, W., Lucas, A., Shippy, R. et al. Evaluation of external RNA controls for the assessment of microarray performance. Nat Biotechnol 24, 1132–1139 (2006). https://doi.org/10.1038/nbt1237
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