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

Petersen D, Chandramouli GV, Geoghegan J, Hilburn J, Paarlberg J, Kim CH, Munroe D, Gangi L, Han J, Puri R, Staudt L, Weinstein J, Barrett JC, Green J, Kawasaki ES - BMC Genomics (2005)

Bottom Line: When concordance was measured for expression ratios significant at p-values of <0.05 and at expression threshold levels of 1.5 and 2-fold, the agreement among the platforms was very high, ranging from 93% to 100%.Our results indicate that the long oligonucleotide platform is highly suitable for expression analysis and compares favorably with the cDNA and short oligonucleotide varieties.All three platforms can give similar and reproducible results if the criterion is the direction of change in gene expression and minimal emphasis is placed on the magnitude of change.

View Article: PubMed Central - HTML - PubMed

Affiliation: Advanced Technology Center, Center for Cancer Research, National Cancer Institute, Gaithersburg, MD 20877, USA. petersed@mail.nih.gov

ABSTRACT

Background: Microarrays for the analysis of gene expression are of three different types: short oligonucleotide (25-30 base), long oligonucleotide (50-80 base), and cDNA (highly variable in length). The short oligonucleotide and cDNA arrays have been the mainstay of expression analysis to date, but long oligonucleotide platforms are gaining in popularity and will probably replace cDNA arrays. As part of a validation study for the long oligonucleotide arrays, we compared and contrasted expression profiles from the three formats, testing RNA from six different cell lines against a universal reference standard.

Results: The three platforms had 6430 genes in common. In general, correlation of gene expression levels across the platforms was good when defined by concordance in the direction of expression difference (upregulation or downregulation), scatter plot analysis, principal component analysis, cell line correlation or quantitative RT-PCR. The overall correlations (r values) between platforms were in the range 0.7 to 0.8, as determined by analysis of scatter plots. When concordance was measured for expression ratios significant at p-values of <0.05 and at expression threshold levels of 1.5 and 2-fold, the agreement among the platforms was very high, ranging from 93% to 100%.

Conclusion: Our results indicate that the long oligonucleotide platform is highly suitable for expression analysis and compares favorably with the cDNA and short oligonucleotide varieties. All three platforms can give similar and reproducible results if the criterion is the direction of change in gene expression and minimal emphasis is placed on the magnitude of change.

Show MeSH
a-b. Clustered image maps showing patterns of expression relationship among genes, platforms, and cell lines. The axes were ordered by hierarchical clustering using an uncentered correlation and the average linkage algorithm for 909 genes expressed at a two-fold or greater level in at least two of the six cell lines. (a) Clustering of all 909 genes (b) A subcluster of 41 genes to show correct clustering and congruence of expression values. As indicated by the cluster trees, all three platforms gave essentially the same relationships among the six cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1140753&req=5

Figure 5: a-b. Clustered image maps showing patterns of expression relationship among genes, platforms, and cell lines. The axes were ordered by hierarchical clustering using an uncentered correlation and the average linkage algorithm for 909 genes expressed at a two-fold or greater level in at least two of the six cell lines. (a) Clustering of all 909 genes (b) A subcluster of 41 genes to show correct clustering and congruence of expression values. As indicated by the cluster trees, all three platforms gave essentially the same relationships among the six cell lines.

Mentions: 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.


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

Petersen D, Chandramouli GV, Geoghegan J, Hilburn J, Paarlberg J, Kim CH, Munroe D, Gangi L, Han J, Puri R, Staudt L, Weinstein J, Barrett JC, Green J, Kawasaki ES - BMC Genomics (2005)

a-b. Clustered image maps showing patterns of expression relationship among genes, platforms, and cell lines. The axes were ordered by hierarchical clustering using an uncentered correlation and the average linkage algorithm for 909 genes expressed at a two-fold or greater level in at least two of the six cell lines. (a) Clustering of all 909 genes (b) A subcluster of 41 genes to show correct clustering and congruence of expression values. As indicated by the cluster trees, all three platforms gave essentially the same relationships among the six cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1140753&req=5

Figure 5: a-b. Clustered image maps showing patterns of expression relationship among genes, platforms, and cell lines. The axes were ordered by hierarchical clustering using an uncentered correlation and the average linkage algorithm for 909 genes expressed at a two-fold or greater level in at least two of the six cell lines. (a) Clustering of all 909 genes (b) A subcluster of 41 genes to show correct clustering and congruence of expression values. As indicated by the cluster trees, all three platforms gave essentially the same relationships among the six cell lines.
Mentions: 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.

Bottom Line: When concordance was measured for expression ratios significant at p-values of <0.05 and at expression threshold levels of 1.5 and 2-fold, the agreement among the platforms was very high, ranging from 93% to 100%.Our results indicate that the long oligonucleotide platform is highly suitable for expression analysis and compares favorably with the cDNA and short oligonucleotide varieties.All three platforms can give similar and reproducible results if the criterion is the direction of change in gene expression and minimal emphasis is placed on the magnitude of change.

View Article: PubMed Central - HTML - PubMed

Affiliation: Advanced Technology Center, Center for Cancer Research, National Cancer Institute, Gaithersburg, MD 20877, USA. petersed@mail.nih.gov

ABSTRACT

Background: Microarrays for the analysis of gene expression are of three different types: short oligonucleotide (25-30 base), long oligonucleotide (50-80 base), and cDNA (highly variable in length). The short oligonucleotide and cDNA arrays have been the mainstay of expression analysis to date, but long oligonucleotide platforms are gaining in popularity and will probably replace cDNA arrays. As part of a validation study for the long oligonucleotide arrays, we compared and contrasted expression profiles from the three formats, testing RNA from six different cell lines against a universal reference standard.

Results: The three platforms had 6430 genes in common. In general, correlation of gene expression levels across the platforms was good when defined by concordance in the direction of expression difference (upregulation or downregulation), scatter plot analysis, principal component analysis, cell line correlation or quantitative RT-PCR. The overall correlations (r values) between platforms were in the range 0.7 to 0.8, as determined by analysis of scatter plots. When concordance was measured for expression ratios significant at p-values of <0.05 and at expression threshold levels of 1.5 and 2-fold, the agreement among the platforms was very high, ranging from 93% to 100%.

Conclusion: Our results indicate that the long oligonucleotide platform is highly suitable for expression analysis and compares favorably with the cDNA and short oligonucleotide varieties. All three platforms can give similar and reproducible results if the criterion is the direction of change in gene expression and minimal emphasis is placed on the magnitude of change.

Show MeSH