Nucleic Acids Research, 2002, Vol. 30, No. 10 e48
© 2002 Oxford University Press
Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays
1Department of Neurology, 2Fishberg Center for Neurobiology and 3Department of Pharmacology, Mount Sinai School of Medicine, New York, NY 10029, USA
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 <250-fold by QRTPCR and could be corrected by the calibration function Fc = Fa(cDNA)q, where Fa(cDNA) is the microarray-determined fold-change comparing experimental with control samples, q is the correction factor and Fc is the calibrated value. The bias observed with the commercial oligonucleotide arrays was less predictable and calibration was unfeasible. Following calibration, fold-change measurements generated by custom cDNA arrays were more accurate than those obtained by commercial oligonucleotide arrays. Our study demonstrates systematic bias of microarray measurements and identifies a calibration function that improves the accuracy of cDNA array data.
* To whom correspondence should be addressed at: Neurology Box 1137, Mount Sinai School of Medicine, New York, NY 10029, USA. Tel: +1 212 241 7075; Fax: +1 212 289 4107; Email: stuart.sealfon{at}mssm.edu The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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C. S. Moreno, S. Ramachandran, D. G. Ashby, N. Laycock, C. A. Plattner, W. Chen, W. C. Hahn, and D. C. Pallas Signaling and Transcriptional Changes Critical for Transformation of Human Cells by Simian Virus 40 Small Tumor Antigen or Protein Phosphatase 2A B56{gamma} Knockdown Cancer Res., October 1, 2004; 64(19): 6978 - 6988. [Abstract] [Full Text] [PDF]
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C. L. Yauk, M. L. Berndt, A. Williams, and G. R. Douglas Comprehensive comparison of six microarray technologies Nucleic Acids Res., August 27, 2004; 32(15): e124 - e124. [Abstract] [Full Text] [PDF]
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V. J. Denef, J. Park, T. V. Tsoi, J.-M. Rouillard, H. Zhang, J. A. Wibbenmeyer, W. Verstraete, E. Gulari, S. A. Hashsham, and J. M. Tiedje Biphenyl and Benzoate Metabolism in a Genomic Context: Outlining Genome-Wide Metabolic Networks in Burkholderia xenovorans LB400 Appl. Envir. Microbiol., August 1, 2004; 70(8): 4961 - 4970. [Abstract] [Full Text] [PDF]
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D. R. Boverhof, K. C. Fertuck, L. D. Burgoon, J. E. Eckel, C. Gennings, and T. R. Zacharewski Temporal- and dose-dependent hepatic gene expression changes in immature ovariectomized mice following exposure to ethynyl estradiol Carcinogenesis, July 1, 2004; 25(7): 1277 - 1291. [Abstract] [Full Text] [PDF]
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V. D. Nair, T. Yuen, C. W. Olanow, and S. C. Sealfon Early Single Cell Bifurcation of Pro- and Antiapoptotic States during Oxidative Stress J. Biol. Chem., June 25, 2004; 279(26): 27494 - 27501. [Abstract] [Full Text] [PDF]
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H. K. Lee, A. K. Hsu, J. Sajdak, J. Qin, and P. Pavlidis Coexpression Analysis of Human Genes Across Many Microarray Data Sets Genome Res., June 1, 2004; 14(6): 1085 - 1094. [Abstract] [Full Text] [PDF]
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B. C. Meyers, D. W. Galbraith, T. Nelson, and V. Agrawal Methods for Transcriptional Profiling in Plants. Be Fruitful and Replicate Plant Physiology, June 1, 2004; 135(2): 637 - 652. [Full Text] [PDF]
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B. H. Mecham, G. T. Klus, J. Strovel, M. Augustus, D. Byrne, P. Bozso, D. Z. Wetmore, T. J. Mariani, I. S. Kohane, and Z. Szallasi Sequence-matched probes produce increased cross-platform consistency and more reproducible biological results in microarray-based gene expression measurements Nucleic Acids Res., May 25, 2004; 32(9): e74 - e74. [Abstract] [Full Text] [PDF]
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J.-B. Fan, J. M. Yeakley, M. Bibikova, E. Chudin, E. Wickham, J. Chen, D. Doucet, P. Rigault, B. Zhang, R. Shen, et al. A Versatile Assay for High-Throughput Gene Expression Profiling on Universal Array Matrices Genome Res., May 1, 2004; 14(5): 878 - 885. [Abstract] [Full Text] [PDF]
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I. S. Lossos, D. K. Czerwinski, A. A. Alizadeh, M. A. Wechser, R. Tibshirani, D. Botstein, and R. Levy Prediction of Survival in Diffuse Large-B-Cell Lymphoma Based on the Expression of Six Genes N. Engl. J. Med., April 29, 2004; 350(18): 1828 - 1837. [Abstract] [Full Text] [PDF]
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P. Yang, O. Agapova, A. Parker, W. Shannon, P. Pecen, J. Duncan, M. Salvador-Silva, and M. R. Hernandez DNA microarray analysis of gene expression in human optic nerve head astrocytes in response to hydrostatic pressure Physiol Genomics, April 13, 2004; 17(2): 157 - 169. [Abstract] [Full Text] [PDF]
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X. Liu and P. De Wulf Probing the ArcA-P Modulon of Escherichia coli by Whole Genome Transcriptional Analysis and Sequence Recognition Profiling J. Biol. Chem., March 26, 2004; 279(13): 12588 - 12597. [Abstract] [Full Text] [PDF]
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D. M. Mutch, P. Anderle, M. Fiaux, R. Mansourian, K. Vidal, W. Wahli, G. Williamson, and M.-A. Roberts Regional variations in ABC transporter expression along the mouse intestinal tract Physiol Genomics, March 12, 2004; 17(1): 11 - 20. [Abstract] [Full Text] [PDF]
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N. Mah, A. Thelin, T. Lu, S. Nikolaus, T. Kuhbacher, Y. Gurbuz, H. Eickhoff, G. Kloppel, H. Lehrach, B. Mellgard, et al. A comparison of oligonucleotide and cDNA-based microarray systems Physiol Genomics, February 13, 2004; 16(3): 361 - 370. [Abstract] [Full Text] [PDF]
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P. Sartipy and D. J. Loskutoff Expression Profiling Identifies Genes That Continue to Respond to Insulin in Adipocytes Made Insulin-resistant by Treatment with Tumor Necrosis Factor-{alpha} J. Biol. Chem., December 26, 2003; 278(52): 52298 - 52306. [Abstract] [Full Text] [PDF]
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I. Chowers, D. Liu, R. H. Farkas, T. L. Gunatilaka, A. S. Hackam, S. L. Bernstein, P. A. Campochiaro, G. Parmigiani, and D. J. Zack Gene expression variation in the adult human retina Hum. Mol. Genet., November 15, 2003; 12(22): 2881 - 2893. [Abstract] [Full Text] [PDF]
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J. Gonzalez-Maeso, T. Yuen, B. J. Ebersole, E. Wurmbach, A. Lira, M. Zhou, N. Weisstaub, R. Hen, J. A. Gingrich, and S. C. Sealfon Transcriptome Fingerprints Distinguish Hallucinogenic and Nonhallucinogenic 5-Hydroxytryptamine 2A Receptor Agonist Effects in Mouse Somatosensory Cortex J. Neurosci., October 1, 2003; 23(26): 8836 - 8843. [Abstract] [Full Text] [PDF]
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K. Korn, P. Gardellin, B. Liao, M. Amacker, A. Bergstrom, H. Bjorkman, A. Camacho, S. Dorhofer, K. Dorre, J. Enstrom, et al. Gene expression analysis using single molecule detection Nucleic Acids Res., August 15, 2003; 31(16): e89 - e89. [Abstract] [Full Text] [PDF]
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Y. Chen, S. Park, Y. Li, E. Missov, M. Hou, X. Han, J. L. Hall, L. W. Miller, and R. J. Bache Alterations of gene expression in failing myocardium following left ventricular assist device support Physiol Genomics, August 15, 2003; 14(3): 251 - 260. [Abstract] [Full Text] [PDF]
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A. Barczak, M. W. Rodriguez, K. Hanspers, L. L. Koth, Y. C. Tai, B. M. Bolstad, T. P. Speed, and D. J. Erle Spotted Long Oligonucleotide Arrays for Human Gene Expression Analysis Genome Res., July 1, 2003; 13(7): 1775 - 1785. [Abstract] [Full Text] [PDF]
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D. P. Dittmer Transcription Profile of Kaposi's Sarcoma-associated Herpesvirus in Primary Kaposi's Sarcoma Lesions as Determined by Real-Time PCR Arrays Cancer Res., May 1, 2003; 63(9): 2010 - 2015. [Abstract] [Full Text] [PDF]
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J. M. Lage, J. H. Leamon, T. Pejovic, S. Hamann, M. Lacey, D. Dillon, R. Segraves, B. Vossbrinck, A. Gonzalez, D. Pinkel, et al. Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array-CGH Genome Res., February 1, 2003; 13(2): 294 - 307. [Abstract] [Full Text] [PDF]
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