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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
Quantitative real-time RT-PCR analysis of 12 genes matched for direction of expression relative to the reference RNA for all three platforms. Log2 ratios are given in table below the graph. This example is a comparison between LnCaP and MCF-10A.
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Figure 6: Quantitative real-time RT-PCR analysis of 12 genes matched for direction of expression relative to the reference RNA for all three platforms. Log2 ratios are given in table below the graph. This example is a comparison between LnCaP and MCF-10A.

Mentions: 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.


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)

Quantitative real-time RT-PCR analysis of 12 genes matched for direction of expression relative to the reference RNA for all three platforms. Log2 ratios are given in table below the graph. This example is a comparison between LnCaP and MCF-10A.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Quantitative real-time RT-PCR analysis of 12 genes matched for direction of expression relative to the reference RNA for all three platforms. Log2 ratios are given in table below the graph. This example is a comparison between LnCaP and MCF-10A.
Mentions: 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.

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