Cited by 520Gene expression and cancer classificationMolecular Biology Techniques and ApplicationsGenomics and Phylogenetic Studies

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Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays

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Yuen, T.·2002·Nucleic Acids Research

DOI 10.1093/nar/30.10.e48PMC PMC115302

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Abstract

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 F(c) = F(a(cDNA))(q), where F(a(cDNA)) is the microarray-determined fold-change comparing experimental with control samples, q is the correction factor and F(c) 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.

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Authors: Yuen, T.
Affiliations: Icahn School of Medicine at Mount Sinai (US)
Corresponding author: Yuen, T.
ORCID: Yuen, T.
Venue: Nucleic Acids Research
Publisher: Oxford University Press (OUP)
Published: 2002-05-15
Volume: 30
Issue: 10
Pages: 48e-48
ISSN: 0305-1048 (ISSN-L)
1362-4962
DOI: 10.1093/nar/30.10.e48
PMC: PMC115302
OpenAlex: W2098453858
Cited by: 520
References: 20
Citations by year: 2026: 1 · 2025: 2 · 2023: 7 · 2022: 1 · 2020: 1 · 2019: 2 · 2018: 3 · 2017: 5 · 2016: 11 · 2015: 5 · 2014: 12 · 2013: 15 · 2012: 16
FWCI: 22.54
Type: journal article
Language: en
Open access: bronze
doi.org
Locations: Nucleic Acids Research (publishedVersion)
Europe PMC (PubMed Central) (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Funders: National Institutes of Health (DK46943, RO1 DK46943)
National Institute on Drug Abuse (P01DA012923, P01-DA12923)
National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK 46943., R01DK046943)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
Keywords: oligonucleotide microarrays, cDNA microarray, measurement bias, gene expression profiling, Affymetrix GeneChip, calibration function, mRNA expression
MeSH terms: Animals, Calibration, Cell Line, Gene Expression Regulation, RNA, Sensitivity and Specificity, Transcription, Genetic, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, Oligonucleotide Array Sequence Analysis

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Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays

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