Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays

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.

Digital quantitative measurements of gene expression

“…(4) GAL gene-GAL1, GAL2, GAL3, GAL7, GAL10, and GAL80-showed a significantly higher expression in galactose than in glucose. The differential expression of the GAL genes between the two growth conditions shows a much higher range of detection than obtained by the microarray hybridization, which is consistent with the literature (Yuen et al, 2002). This further validates the sensitivity and the range of detection of the DAGE approach.…”

“…This is in general agreement with the relative expression level changes determined from microarray data (Dudley et al, 2002). Note also that the DAGE technique has a larger dynamic range than do other expression profiling techniques (Yuen et al, 2002). Namely, we were able to quantitatively measure gene expression at very low levels with similar precision as to measurements at very high expression levels; furthermore, the upper limit of detection is unbounded because one can always increase the dilution an unlimited number of times.…”

A cluster of differentially expressed signal transduction genes identified by microarray analysis in a rat genetic model of alcoholism

“…The major phenotypic differences between the rat lines seem to be encoded by modest differences in brain gene expression, although the microarray analysis may underestimate their magnitude. 35 Quantitative real-time RT-PCR confirmation of differential preproNPY expression indicates that differences of this magnitude can be detected by the array analysis using the present experimental conditions, and, perhaps more importantly, the chosen statistical model. We conclude that gene expression profiling with oligonucleotide microarrays is a feasible strategy to detect previously unknown differences in basal gene expression between genetically determined phenotypes.…”

Gene Expression of Metabolic Enzymes and a Protease Inhibitor in the Prefrontal Cortex Are Decreased in Schizophrenia

“…Therefore, Q-PCR is a reliable and sensitive method for confirmation of microarray findings. The microarray studies, whether using a commercial oligonucleotide platform or cDNA array, consistently underestimate the gene expression fold changes when compared to Q-PCR method (13). Only one prior microarray report of schizophrenia has reported real-time Q-PCR gene expression results (14).…”