Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays
Open accessAbstract
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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A robust global normalization algorithm, using expression levels of the housekeeping genes ribosomal protein S11 (rps11), β-actin (actb), and α-tubulin (tuba), was used for all experiments.
“A robust global normalization algorithm, using expression levels of the housekeeping genes ribosomal protein S11 (rps11), -actin (actb), and ␣-tubulin (tuba), was used for all experiments, as described elsewhere (15,16).”
From Cytokine Response Is Determined by Duration of Receptor and Signal Transducers and Activators of Transcription 3 (STAT3) Activation (2013) · cites this paper as [16]
The cDNA array calibration section describes a power-law correction fitted to microarray and PCR measurements, not normalization using the three named housekeeping genes. The Microarray and QRTPCR reference measurements section mentions beta-actin as one gene measured for reproducibility, but does not describe its use with rps11 and tuba in a global normalization algorithm. Searches for the named genes and the normalization method found no supporting passage; page numbers were unavailable in our copy. (LLM, conf=0.76)
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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
- 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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