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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.

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a-f. Quantitative RT-PCR analysis of 10 mismatched genes in the six cells lines for all three platforms. (a) MCF10A, (b) LnCaP, (c) OCI-Ly3, (d) Jurkat, (e) SUDHL-6 and (f) L428.
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Figure 7: a-f. Quantitative RT-PCR analysis of 10 mismatched genes in the six cells lines for all three platforms. (a) MCF10A, (b) LnCaP, (c) OCI-Ly3, (d) Jurkat, (e) SUDHL-6 and (f) L428.

Mentions: 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.


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-f. Quantitative RT-PCR analysis of 10 mismatched genes in the six cells lines for all three platforms. (a) MCF10A, (b) LnCaP, (c) OCI-Ly3, (d) Jurkat, (e) SUDHL-6 and (f) L428.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: a-f. Quantitative RT-PCR analysis of 10 mismatched genes in the six cells lines for all three platforms. (a) MCF10A, (b) LnCaP, (c) OCI-Ly3, (d) Jurkat, (e) SUDHL-6 and (f) L428.
Mentions: 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.

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