Three microarray platforms: an analysis of their concordance in profiling gene expression

Genes in common across platforms

Only genes common to all three platforms were used in the comparison. Genes were matched by UniGene Cluster (UniGene Build #161), and unique cluster memberships were determined for each array type, as listed in Table 1 and enumerated in the Venn diagram in Figure 1. The intersection for the three platforms consisted of 6430 UniGene clusters, and all analyses were carried out with all of these genes or a subset of them.

	Incyte UniGEM2	Hs-Operon V2	HG-U133A
Total features / probe sets:	9128	21522	22215
Distinct UniGene clusters:	8097	19179	13899
	UniGem2 & Operon V2	UniGEM2 & HG-U133A	Operon V2 & HG-U133A
Genes in common	7082	6593	12999
Genes in common in all arrays	6430	6430	6430

Comparison of expression ratios

Correlation between platforms

The scatter plots shown in Figures 2a–c indicate graphically the correlation between the platforms for Jurkat RNA. Values of r for all platforms and all cell lines are given in Table 4. The values ranged from 0.7 to 0.8. Operon and Affymetrix were better correlated than were Operon and Incyte. The values were intermediate for the relationship between Incyte and Affymetrix. Figures 2d–f and Table 5 indicate that the correlation coefficients between replicates within the same platform are closer, as expected, when compared to the correlations between platforms. In general, however, there was reasonably good concordance among the platforms.

Platform	Jurkat	L428	SUDHL	OCI-Ly3	LNCaP	MCF10A	Median
Incyte/Operon	0.727	0.707	0.708	0.724	0.710	0.708	0.709
Incyte/Affy	0.767	0.777	0.781	0.780	0.741	0.744	0.772
Operon/Affy	0.813	0.784	0.783	0.790	0.796	0.782	0.787

Platform	Jurkat	L428	SUDHL	OCI-Ly3	LNCaP	MCF10A
GEM2	0.859	0.919	0.902	0.873	0.827	0.845
Operon V2	0.916	0.805	0.849	0.828	0.951	0.928
HG-U133A	0.956	0.957	0.960	0.952	0.963	0.954

Principal Component Analysis (PCA)

A projection on the first three principal components, which together explain 48.8 % (21%, 14%, and 13%) of the total variance, is shown in Figure 3. Close clustering of the cell samples is observed in this projection, indicating appreciable agreement among array platforms.

"Correlation of Correlations" analysis

The global concordance of the three platforms across all of the cell lines was estimated by calculating the "correlation of correlations" coefficient [10, 11]. As seen in Figure 4a–c, the correlations for the three platforms across all cells lines were quite good. The Pearson correlation of correlation coefficients was 0.965 between Operon and Incyte, 0.995 between Affymetrix and Incyte, and 0.956 between Operon and Affymetrix.

Clustered Image Map (CIM) visualization and analysis [12]

We used hierarchical clustering to demonstrate graphically the relationships among platforms, among cell lines, and among genes. 909 genes expressed at two times background or more in at least two of the six cell lines were included in the analysis. The resulting CIM ("heat map") is shown in Figure 5a–b. All three platforms cluster together for all six cell lines, as one would wish to find, and almost all of the gene expression values show reasonable concordance. Only a few exceptions can be seen in the cluster shown in Figure 5b. Some of the mismatches may be due to simple errors in gene identification, rather than to the technologies of the platforms. The Incyte library is guaranteed by the manufacturer to be only 90% correct, and an unknown percentage of the Operon and Affymetrix oligonucleotides may have been designed on the basis of incorrect sequences in the public databases. Indeed, we found one oligonucleotide in the Operon set that was apparently designed from an EST sequence that has since been withdrawn from the UniGene database (see RT-PCR studies below). In any case, the concordance is quite high across all platforms with this method of analysis as well as with the others.

Quantitative real-time RT-PCR

In a pilot study with the three platforms, we compared and contrasted gene expression values for only the cell lines MCF10A and LNCaP. RT-PCR data for twelve genes are shown in Figure 6. Most of the values are in reasonable agreement except that there are differences in the magnitudes of the expression ratios. As found in other studies, the RT-PCR values are generally higher, probably because ratios are "flattened" with the microarray platforms. Affymetrix ratios are sometimes higher, but that may simply reflect the method of quantitation used in their analysis. The cDNA array ratios are generally lower than those from other platforms. Because the cDNA fragments are longer and double-stranded, they are more prone to non-specific hybridization and can cross-hybridize more easily to related sequences. These characteristics of the probes may result in higher background signal and concomitant reduction in dynamic range of the ratios. In general, we have found that the long oligonucleotide arrays have a larger dynamic range than the do the cDNA arrays.

For further RT-PCR analysis, we chose a set of ten genes to test the accuracy of the three array platforms for all six cell lines. Those genes were chosen because there appeared to be a discrepancy among platforms in the direction of their ratios (i.e., whether they ratios were greater or less than unity). The results are shown in Figures 7a–f. Of special interest was gene ETR101, in which the Operon array was in disagreement across all cell lines. Further inquiry revealed that the sequence had been found to be incorrect and had been removed from the UniGene database. Since the oligonucleotide had been designed from the incorrect sequence, it is not surprising that it gave a different value. Other discrepancies may be due to similar sequence errors, as even the most up-to-date databases are still being corrected and modified. In the case of AGL, the RT-PCR assay is in disagreement in several cases with two out of three of the array platforms; it appears to demonstrate an upregulation of the mRNA, whereas the arrays, with the exception of Incyte, point to a downregulation. Although RT-PCR is supposed to be the "gold standard" for measuring gene expression, this result shows that caution is indicated in interpreting results with even the PCR technology.

The same RNA templates should be used throughout the entire experiment

RNA preparations from similar, but non-identical, biopsy samples can yield dissimilar results [35], and RNA from different versions of the same cell line can yield widely different expression profiles [36, 37].

Only genes common to all platforms should be used in the analysis

That may seem an obvious requirement, but it is not always easy to meet in practice. For example, careful study of sequences present in various cDNA arrays indicates that 20–30% of spots may contain the wrong clone or a misidentified one [10, 14, 38–40]. A similar situation exists for the short oligonucleotide platform (Affymetrix) because more than 19% of the sequences have been reported not to correspond to the appropriate mRNA Reference Sequence [41, 42]. In these studies, measurements of cross-platform consistency were significantly improved when only sequence-matched genes were used. Similarly, discrepant results in studies using cDNA and Affymetrix platforms could be explained by errors in gene sequences [43]. The percentage of incorrect or misidentified sequences in the long oligonucleotide libraries is unknown, but we appear to have found one sequence in error during our own RT-PCR studies. Overall, then, significant disagreement in expression ratios among platforms may be due to sequence errors, not to intrinsic differences among array systems.

Only spots with signals significantly above background (and that pass stringent quality-control filters) should be used in the comparisons

With most high-density arrays, a large proportion of the genes can be expected to have signals near background, as probably fewer than half of all human transcripts are expressed in any particular cell type or tissue [44]. Although stringent filtering decreases the number of measurements included in the analysis, the outcome will be more meaningful.

When comparing results from the three platforms, the magnitudes of ratios were often quite different, but there was generally good agreement in the direction of differences in expression (Figs. 6 &7, Tables 2 &3). Thus, it may be less productive to correlate absolute changes in expression than it is to look for agreement in direction.

Overall, the two oligonucleotide platforms were the most concordant pair. Possible reasons are (i) that the array targets are single stranded, all of the same size (25-mer or 70-mer), with approximately the same melting temperature; (ii) the array elements have a much higher molarity of gene-specific sequences than do the cDNA arrays; (iii) that oligonucleotides tend to be more specific in terms of sequence and less prone to cross-hybridization than are cDNAs.

Experimental design

Three array platforms were tested. Incyte cDNA arrays and Operon long oligonucleotide (70-mer) arrays were printed in house, and Affymetrix 25-mer HG U133A arrays were purchased from the manufacturer. The cDNA and long oligonucleotide arrays were assayed in quadruplicate, two each of red/green and green/red for dye "flips" (reverse fluor experiments) to help eliminate dye bias (except for L428 that had 3 replicates and OCI-Ly3 with 5 replicates). The Affymetrix arrays were tested in duplicate. RNA preparations from six cell lines were tested with each platform using a universal reference RNA (Stratagene) as the standard.

Cell lines and RNAs

Cell lines were grown and RNA isolated at the core Gene Expression Laboratory, NCI-Frederick. MCF10A (benign mammary epithelial), LNCaP (prostate carcinoma), Jurkat (T-cell lymphoma), SUDHL6 (germinal center B-cell like diffuse large B-cell lymphoma), OCI-Ly3 (activated B-cell like diffuse rare B-cell lymphoma and L428 (Hodgkin's lymphoma) were grown under standard conditions [55], and RNA was isolated from the cells using TriReagent following the manufacturer's protocol (Molecular Research Center, Inc., Cincinnati, OH). Integrity of the RNA was confirmed by analysis with the Agilent 2100 Bioanalyzer (Palo Alto, CA) using the RNA 6000 LabChip^® kit. For use as the index standard, Human Universal Reference RNA (HUR RNA) was purchased from Stratagene (La Jolla, CA).

Preparation of cDNA and oligonucleotide arrays

Arrays with 10,000 cDNAs were prepared from ready-to-print UniGEM2 libraries obtained from Incyte, Inc. (Wilmington, DE). Version 2.0 libraries containing 22,000 oligonucleotides of 70 bases in length were obtained from Operon, Inc. (Alameda, CA). Arrays were printed by standard protocols on Corning Ultra-GAPS II slides (Corning, NY) using a GeneMachine^® (San Carlos, CA) instrument. cDNAs were suspended at a concentration of 100 μg/ml and oligonucleotides at 25 μM in 3XSSC buffer, and the arrays were printed using SMP3 pins from Telechem International (Sunnyvale, CA). The spotted nucleic acids were fixed to the slides using protocols supplied by the manufacturers. The 25-mer oligonucleotide HG U133A Genechip^® arrays were purchased from Affymetrix, Inc (Santa Clara, CA).

Labeling and purification of targets

Labeled cDNAs from cell samples for hybridization to the long-oligonucleotide and cDNA arrays were synthesized and labeled by the indirect amino-allyl method, using reagents and protocols supplied by the manufacturer. For cDNA synthesis, we used Stratascript reagents (Stratagene, La Jolla, CA) and Cy3 and Cy5 fluorophore amino-allyl reagents from Amersham (Piscataway, NJ). Twenty micrograms of total RNA was used for each synthesis. Labeled cDNA targets were purified using Minelute purification kits (Qiagen, Valencia, CA). cRNA targets for the Affymetrix arrays were synthesized, labeled, and purified according to vendor's (Affymetrix) instructions. Briefly, 10 μg of total RNA was used to make double-stranded cDNA using reagents and protocols obtained from Invitrogen (Carlsbad, CA). Linear amplification was carried out by a modification of the Eberwine T7 method [56], and biotin was incorporated into the cRNA using the Enzo High Yield RNA Transcript labeling Kit (EnzoDiagnostics, Farmingdale, NY).

Hybridization and washing of arrays

The cDNA and long-oligonucleotide microarrays were prehybridized in 40 μl of 5XSSC with 0.1% SDS and 1% BSA at 42°C for 30 minutes. The prehybridization solution was removed, and arrays were hybridized for 16 hours at 42°C in 5XSSC buffer containing Cy3/Cy5 labeled targets, 25% formamide, 0.1% SDS, 1 μg Cot-1 DNA, and 1 μg poly A RNA. The cDNA arrays were washed at room temperature in 2XSSC, 0.1% SDS for 2 minutes, 1XSSC for 2 minutes, 0.2XSSC for 2 minutes and 0.05XSSC for 1 minute. The long-oligonucleotide arrays were treated the same except that the last wash step was omitted. The slides were dried by centrifugation at 650 rpm for 3 minutes. The Affymetrix arrays were hybridized and washed using the manufacturer's protocol. The arrays were then stained with streptavidin-phycoerythrin using the standard antibody amplification protocol (GeneChip^® Expression Manual, Affymetrix, Inc., Santa Clara, CA).

Array scanning and image processing

Long-oligonucleotide and cDNA arrays were scanned using an Axon 4000B scanner at 10-micron resolution. Images were processed, and signals from spotted arrays were quantitated using Genepix 3.0 software (Axon Instruments, Union City, CA). The Genepix result files, including signal, background, standard deviation, pixel statistics and quality parameters for both channels were deposited in the microarray database (mAdb) maintained by the NCI/CIT bioinformatics group [57]. The data were filtered on the basis of signal levels and spot quality. Local background values were subtracted from spot intensities to obtain signal values. Data were included if the signal-to-background ratio was ≥ 2, the signal intensity was >100, the spot diameter was between 50 and 180 microns, at least 70% of the pixels were above their standard deviation and the spot was not flagged as "bad" visually. Arrays were normalized by median-centering the logarithmic ratios so that the median ratio of all genes that passed through the filters was equal to 1. For cDNA arrays, normalized expression ratios of 9050 genes were calculated, and the same procedure was applied to long-oligonucleotide arrays for the expression of 20,799 genes. Affymetrix HG-U133A arrays were scanned with the Affymetrix GeneArray scanner at 488 nm and 3-micron resolution. The images were analyzed using Microarray Suite 5.0 software (MAS5; Affymetrix Inc., Santa Clara, CA). Cell-line to HUR expression ratios were computed by comparative analysis of MAS5 values. The data were filtered using MAS5's signal detection and change calls generated at recommended default settings. The ratios included were those that had present calls for signal detection or an increase or decrease call associated with the ratio. The filtered data contained 17,647 genes. For all statistical calculations, logarithmic values of ratios to the base 2 were used.

Determination of genes in common among all platforms

Genes were matched by UniGene cluster methods [11, 58], and expression levels were compared for only the 6,430 genes common to all platforms (Table 1 and Figure 1). UniGene clusters (Homo sapiens: UniGene Build #161) of probes of all three platforms were determined by the NCI/CIT Bioinformatics group [57] using the BLAST program from the National Center for Biotechnology (NCBI, Bethesda, MD 20894). There were multiple probes for some of the UniGene clusters. This resulted in matching of two or more probes of one platform to one or more probes of another platform. All possible combinations across three platforms were considered for each UniGene cluster. One probe from each of the platforms was selected as follows. Initially, for a given combination, the replicates were averaged to obtain expression patterns of six RNAs on three platforms. The sum correlation coefficients of these three patterns to their mean pattern was determined. The combination having the highest sum was selected for further analysis. This method relies on the assumption that the probes specific for a gene yield similar expression patterns independent of the platform. The matched expression ratios will be made available at our website http://nciarray.nci.nih.gov/publications/.

Estimation of total matched versus mismatched expression values

Expression ratios for genes in common across all cell lines and platforms were determined. If the ratio of a gene was ≥ 1 or ≤ 1 for both platforms being compared, the expression was considered matched irrespective of the magnitude. Otherwise, the ratios were considered to be mismatched (i.e., in opposite directions). These values give a rough, binary indication of the correlation between platforms (Table 2). Concordance between platforms using significant expression ratios at p-value <0.05 and 1.5 to 2-fold threshold levels are given in Table 3.

Correlation of expression among all platforms

To determine how well the data from the three platforms coincided, correlation coefficients were obtained from analysis of scatter plots of the mean expression values from the three array formats. Figure 2a–c are examples of scatter plot analyses of the three platforms using data from the Jurkat cell line RNA. The boundaries for ratio values greater than two-fold are delineated by the external lines. Correlation coefficients for all three pairs of platforms for all six cell lines are listed in Tables 4 &5).

Principal Component Analysis (PCA)

The global gene expression patterns of the six cell lines in all three platforms were studied by principal component analysis (PCA) [59]. All genes (3186) with signals above background were included. Differences in signal magnitude among platforms were nulled out by normalizing the data from each to unit standard deviation. Individual platform variations were accounted for by employing unit variance. A projection on the first three principal components, which explained 48.8% (21%, 14%, and 13%) of the total variance, is shown in Fig. 3. The calculations were done using Partekpro 5.0 software (Partek Inc., St. Charles, MO)

Platform concordance by "correlation of correlations" coefficient

The global concordance of expression levels of the three platforms can be expressed in terms of the 'correlations of correlations' coefficient described previously [10, 11]. To perform the computations, step 1 was calculation of the Pearson correlation coefficient across all matched genes for each of the 15 possible pairs of cell lines for each platform. Step 2 was calculation of the Pearson correlation coefficients of those correlation coefficients for the three possible ways of pairing the three platforms. The results are shown in Figures 4a–c.

Hierarchical cluster analysis and Clustered Image Maps (CIMs)

Hierarchical clustering of individual replicates (data not shown), including 3662 genes detected in 80% of the arrays revealed a grouping of RNA samples independent of the type of array platform. A set of 909 genes expressed at >two-fold levels in all platforms in at least two cell lines was used for hierarchical cluster analysis to determine how closely the genes, cell lines and platforms corresponded in their expression values. As a distance metric, we used 1-r, where r is the Pearson correlation coefficient [60]. Cluster nodes were determined using an average linkage algorithm. In the resulting CIM (heat map) [14a], up- and down-regulation with respect to the reference RNA are color-coded as red and green, respectively (Figure 5a–b).

Quantitative real-time RT-PCR

RNA preparations from each of the six cell lines and Stratagene Human Universal Reference RNA were converted into single-stranded cDNA using the Applied Biosystems High-Capacity cDNA Archive kit (ABI, Foster City, CA). Primer and probe sets were obtained as ABI Assays-on-Demand™ Gene Expression Products (TaqMAN^® MGB probes, FAM™ dye-labeled) for a set of genes to be studied, as well as GAPDH and BACT, which were used as comparative controls. All quantitative PCR reactions were performed in quadruplicate, with two carried out in an ABI Prism^® 7000 sequence detection system and two in a Corbett Research R-300 instrument (Corbett Research, Sidney, Australia). The results were analyzed using the "Relative Quantitation of Gene Expression" method described in ABI Prism 7700 Sequence Detection System User Bulletin #2, Rev B. An initial study comparing only two cell lines, LnCaP and MCF10A, was carried out for the three platforms. Twelve genes that matched in direction of change were chosen for Q-PCR analysis, and the results are shown in Figure 6. Subsequently, ten genes were chosen for analysis using all six cell lines and three platforms. Genes in that set were deliberately chosen for mismatched ratios to determine if any platform was in error more often than the others. The results are shown in Figures 7a–f.