* School of Pharmacy,
Waisman Center,
Molecular and Environmental Toxicology Center, and
Center for Neuroscience, University of Wisconsin, Madison, Wisconsin 53705
Received May 29, 2002; accepted July 10, 2002
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ABSTRACT |
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Key Words: DNA arrays; gene expression; antioxidant responsive element; cross-hybridization; tert-butylhydroquinone.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Microarray Analysis
cDNA array system.
mRNA was sent to IncyteGenomics for analysis on the Incyte Unigem V2.0 arrays (containing 8556 genes/EST cluster). Since Cy3 and Cy5 have a different labeling efficiency for target cDNAs, we reversed the samples for different labeling each time, to determine the reproducibility of the results. Note that the operators were blind to the specific category to which each sample belonged. Following the hybridization and washing, the relative expression level of both cDNA populations were measured and compared by making the balanced Cy3/Cy5 fluorescence ratio. In this study, genes were considered differentially expressed if the ratio was 1.74-fold, at 99% confidence (http://www.incyte.com). The cDNAs corresponding to genes were sorted by enzyme, function, or pathway cluster analysis using Gemtools software (V2.4.2, IncyteGenomics). Gene group data were exported to MS Excel for further analysis.
Oligonucleotide array system.
The samples analyzed by Incyte were also used to generated fragmented cRNA probes for Affymetrix GeneChip analysis. Briefly, cDNA was synthesized from 500 ng mRNA by using superscript choice kit (GIBCO/BRL) with a T7-(dT)24 primer incorporating a T7 RNA polymerase promoter. The cRNA was prepared and biotin-labeled by in vitro transcription (Enzo Biochem). Labeled cRNA was fragmented by incubation at 94°C for 35 min in the presence of 40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate. Fifteen mg of the fragmented cRNA was hybridized for 16 h at 45°C to a HuGene FL (original version, 6800 genes/EST cluster) or HG U95Av2 array (updated version, 9670 genes/EST clusters). After hybridization, the genechips were automatically washed and stained with streptavidin-phycoerythrin using a fluidics station. Arrays were finally scanned at 3-mm resolution using the Genechip System confocal scanner made for Affymetrix by Aligent. Microarray Suite 4.1 and 5.0 software from Affymetrix were used on HuGene FL and HG U95Av2 arrays, respectively, to determine the relative abundance of each gene, based on the average difference of intensities. Output from the genechip analysis was merged with the Unigene or GenBank descriptor and stored as an Excel data spreadsheet.
RT-PCR
We used RT-PCR to validate the increased mRNA level of nine selected genes. PCR primers specific for the genes of interest were used for cDNA synthesis and amplification as follows. Unique oligonucleotide primer pairs for NQO1, HO1, GR, aldo-keto reductase family 1, -glutamylcysteine ligase regulatory and catalytic subunits (GCLR and GCLC), thioredoxin reductase (TR), neurofilament heavy subunit, and ß-actin were prepared by IDT (Coralville, IA). Total RNA, purified from cell pellets with Trizol Reagent (GIBCO/BRL), was subjected to RT-PCR with Promega Transcription System (Madison, WI). The reaction mix (20 ml) contained 200 mM dNTP, 0.45 mM of each primer, and 1 mg of total RNA and AMV Reverse Transcriptase (15 U). RNA was reverse-transcribed at 42°C for 30 min and DNA was amplified by an initial incubation at 94°C for 4 min, followed by
2535 cycles at 94°C for 0.5 min,
5558°C for 0.6 min, 72°C for 0.5 min, and a final extension at 72°C for 7 min. The PCR products were then separated by electrophoresis in a 1.2% agarose gel and visualized by ethidium bromide staining. The number of cycles and melting temperature were adjusted, depending on the gene being amplified.
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RESULTS |
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Figure 1A illustrates the dramatic increase in the number of publications using microarrays since 1999 (1999, 60 ± 41.6; 2000, 195 ± 34.5; 2001, 627 ± 66.7) and Figure 1B
shows that in the last three years 75% of the arrays used have been provided commercially. Some reasons for the recent increase in microarrray use are certainly the improvement in cost, convenience, availability, and performance of commercial array systems. It should also be noted that either Affymetrix or IncyteGenomics provided the vast majority of commercial arrays used for global screening. Only one-fourth of the publications worked on homemade arrays that usually were designed to meet the specific interest of researchers. This statistical analysis demonstrates the necessity of this study to compare these two global commercial array systems, which constitute over 30% of the total arrays used in the last three years and somewhere between 75 and 90% of the total global screening arrays used.
Distinct Gene Expression Patterns Revealed by the Two Array Systems
Oligonucleotide array system.
The Affymetrix algorithm for detecting differential expression considers several parameters from the raw data, and the results of these analyses are presented in the Supplemental Appendix of Affymetrix Microarray Suite User Guide. "Difference Call" or "Change Call" was used to determine the differential gene expression initially. Original (HuGene FL) and updated versions (HG U95Av2) of human chips were analyzed by Microarray Suite V. 4.1 and V. 5.0, respectively. The data generated from HuGene FL chips has a high correlation with that from the U95Av2 chips. There were dramatic variations in the number of genes called different (I, increased; D, decreased) calculated from the matched sample pairs. For example, the number of I/D genes generated from three pair-matched, 8-h treatment samples on the U95Av2 chips were 173/283, 171/151, and 597/300. We defined increased, decreased, or no change of expression for individual genes based on ranking of the "difference call" or "change call" from the three pair-matched comparisons. Briefly, No change, 0; Marginal increase, 1; Increase, 2; Marginal decrease, 1; and Decrease, 2. The cutoff value for increase/decrease was set as ± 3 because of the marginal calls. This screening process led to the identification of a certain number of genes whose expression levels consistently increased or decreased. Application of these principles to the other sample pairs led to the identification of approximately 218 (2.3% of total) genes on the U95Av2 chip whose expression consistently increased (207) or decreased (11) after tBHQ treatment for 8 h. The number of increased or decreased genes on the HuGene FL and HG U95Av2 chips were calculated and are shown in Figure 2.
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Cross-hybridization.
Sensitivity and specificity are the two major features that researchers are concerned with in performing microarray analysis. Microarray sensitivity is often defined as the minimum reproducible signal (fluorescence intensity) detected by a given array-scanning system. The sensitivity (fluorescence intensity) and specificity (fold) was very different in the same genes when comparing the two platforms. For example, HO1 and NQO1 changed 7.38-fold and 5.40-fold on the U95Av2 chips, respectively, after 8 h of treatment, whereas the cDNA array showed 1.14-fold and 1.04-fold change for both genes (Table 1). Cross-hybridization must be considered since false positive signals sometimes disguise real changes and make the basal expression of individual genes very high. This can lead to a false impression of high sensitivity, albeit this high sensitivity is reproducible. For example, labeling and hybridization of IMR-32 cell cRNA with U95Av2 chips did not result in a detectable signal for some probe sets designed to detect certain phase-II detoxification enzymes such as NQO1 and HO1. Both were called absent in controls (Table 2
). After treatment with tBHQ, there was a significant increase in signal (fluorescence intensity) of these genes and both were called present (Table 2
). The Incyte 2-color analytic strategy provided the relative probe value of gene expression, which seems to be aberrantly high for NQO1 and HO1, both in control and treated groups. Thus, the obvious change in NQO1 based on RT-PCR (Li and Johnson, 2002
), Western blot (Li and Johnson, 2002
), and enzymatic activity (Moehlenkamp and Johnson, 1999
) has been buried in the cross-hybridization signal on the spotted arrays. This holds true for genes whose basal gene expression is low (e.g., HO1) or high (e.g., GCLR; Table 2
). As a result, the disparity in the final datasets probably resulted from some cross-hybridization to the longer cDNA probes on the Unigem V.2 arrays.
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DISCUSSION |
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The key factor in determining the specificity and sensitivity of the two commercial array systems we used was the length of the DNA employed as the probe sequence on the array. For the Incyte long cDNA array, every clone was selected from the UniGem database, which contains sequences that have been verified as representative of the desired gene. The sequence of probes varied from 5005000 base pairs, averaging 1000 bp (http://www.incyte.com) One of the problems associated with having such a large target sequence is that it becomes difficult to control the hybridization efficiencies of various cDNA probes. Since the hybridization conditions are based on the length of nucleic acid fragments and the compositions of G + C and A + T (or A + U), hybridization efficiency can vary widely when long cDNA sequences are used as a probe. In addition, the issue of cross-hybridization of related or overlapping genes is also potentially an important limitation of the cDNA-based microarray, since different genes may encode common domains and can have some degree of sequence identity or homology with proteins from other genes (Evertsz et al., 2001; Kane et al., 2000
). As a result, gene families present a potential problem, because in many cases these genes have a great degree of sequence identity and can only be distinguished from each other by the design and use of gene-specific hybridization probes. In the case of Affymetrixs oligonucleotide array, the 25-oligomer probes are designed to uniquely represent the desired cognate gene through blasting the GenBank database, thus minimizing cross-hybridization between similar sequences (Lipshutz et al., 1995
; Lockhart et al., 1996
). Using oligonucleotides also makes it easier to design and select probes from the same or different genes with similar G + C content and putative melting temperature. Other criteria for the selection of probes include unique sequence, minimum secondary structures, and 3' terminal sequence selection, all of which serve to insure greater specificity in the probe-target binding (http://www.affymetrix.com).
The oligonucleotide approach for the analysis of gene expression has been criticized for a lack of sensitivity (Schulze and Downward, 2001). Since a single capture probe is not always sufficient to distinguish the expression of a particular gene, the use of multiple capture probes to represent a single gene in the Affymetrix array system is intended to avoid the problem of cross-hybridization as well as to increase sensitivity. Thus, one of the likely causes of increased specificity in oligonucleotide arrays, as compared with long cDNA arrays, is a decrease in cross-hybridization with highly homologous genes.
Although it is true that shorter oligonucleotides (15 to 20 mers) promise more specific binding with probes, some papers recently indicated that 50 or 70 mers yield good sensitivity while maintaining the excellent specificity of shorter sequences. Operon Techologies (http://www.westburg.nl) compared the sensitivity of 35, 50, 70, and 90-mers for detecting highly expressed genes and genes expressed at moderate or low levels, and found that the 70-mer lengths performed best. However, the results from MWG Biotech (http://www.mwgbiotech.com) showed that 50-mer oligonucleotide arrays provided high specificity and excellent sensitivity, while 70 mers only increased formation of secondary structure (Kane et al., 2000).
Microarray analysis has gone from an interesting idea to a core technology in just a few years. The recent trend of institutions bringing these tools to core facilities should only further open the way for academic researchers to use this innovation. While only a few of the academic core facilities surveyed offered access to the Affymetrix GeneChips, a much larger number offered cDNA array technology. Also based on the number of publications associated with microarray technology, nearly 75% of papers use spotted cDNA arrays as a means of profiling the gene expression patterns since 1999. In addition, the wide use of the Affymetrix microarray system has been limited, due to the more involved sample processing steps. The investigator is generally responsible for isolating RNA, creating double-stranded cDNA incorporating a T7 promoter, performing IVT to get labeled cRNA, fragmenting the cRNA, and providing QC documentation of these steps. There are also additional costs in that Affymetrix recommends running a test chip to verify the quality of samples.
In the context of a drug-treated cell line presented herein, oligonucleotide arrays give a more accurate and comprehensive scenario of gene-expression profiles, which in turn ensures with a higher probability that further research work will proceed down the correct path. Clearly the recent efforts to genetically profile toxic compounds and promising pharmaceuticals requires the best data sets possible for application to drug discovery. These data strongly imply that oligonucleotide-based arrays are more reliable for global screening compared to long cDNA array at the present time. What the future holds, however, remains to be determined.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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