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The chemiluminescence based Ziplex automated workstation focus array reproduces ovarian cancer Affymetrix GeneChip expression profiles.

Quinn MC, Wilson DJ, Young F, Dempsey AA, Arcand SL, Birch AH, Wojnarowicz PM, Provencher D, Mes-Masson AM, Englert D, Tonin PN - J Transl Med (2009)

Bottom Line: Overall, 75 of 93 (80.6%) genes exhibited consistent results in normal versus tumor tissue comparisons for both platforms (p < 0.001).There was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between tumor versus normal samples (R = 0.93) and by Bland-Altman analysis, where greater than 90% of expression values fell within the 95% limits of agreement.The reproducibility and ease-of-use of the technology suggests that the Ziplex array is a suitable platform for translational research.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Human Genetics, McGill University, Montreal, Canada. michael.quinn@mail.mcgill.ca

ABSTRACT

Background: As gene expression signatures may serve as biomarkers, there is a need to develop technologies based on mRNA expression patterns that are adaptable for translational research. Xceed Molecular has recently developed a Ziplex technology, that can assay for gene expression of a discrete number of genes as a focused array. The present study has evaluated the reproducibility of the Ziplex system as applied to ovarian cancer research of genes shown to exhibit distinct expression profiles initially assessed by Affymetrix GeneChip analyses.

Methods: The new chemiluminescence-based Ziplex gene expression array technology was evaluated for the expression of 93 genes selected based on their Affymetrix GeneChip profiles as applied to ovarian cancer research. Probe design was based on the Affymetrix target sequence that favors the 3' UTR of transcripts in order to maximize reproducibility across platforms. Gene expression analysis was performed using the Ziplex Automated Workstation. Statistical analyses were performed to evaluate reproducibility of both the magnitude of expression and differences between normal and tumor samples by correlation analyses, fold change differences and statistical significance testing.

Results: Expressions of 82 of 93 (88.2%) genes were highly correlated (p < 0.01) in a comparison of the two platforms. Overall, 75 of 93 (80.6%) genes exhibited consistent results in normal versus tumor tissue comparisons for both platforms (p < 0.001). The fold change differences were concordant for 87 of 93 (94%) genes, where there was agreement between the platforms regarding statistical significance for 71 (76%) of 87 genes. There was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between tumor versus normal samples (R = 0.93) and by Bland-Altman analysis, where greater than 90% of expression values fell within the 95% limits of agreement.

Conclusion: Overall concordance of gene expression patterns based on correlations, statistical significance between tumor and normal ovary data, and fold changes was consistent between the Ziplex and Affymetrix platforms. The reproducibility and ease-of-use of the technology suggests that the Ziplex array is a suitable platform for translational research.

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Comparison of the fold change difference in expression between NOSE and TOV samples for the Ziplex and Affymetrix platforms. A: The log2 fold change between the NOSE and TOV samples (mean NOSE signal intensity/mean TOV signal intensity) was calculated for the expression values of all 93 probes and plotted. Linear regression was performed resulting in the following model: log2 Affymetrix NOSE/TOV = 0.180098 + 1.0251794 log2 Ziplex NOSE/TOV with a Pearson's correlation coefficient (R) of 0.93. Probes that were not significant (p > 0.001 based on a Welch Rank Sum test) on either platform are indicated in grey, probes significant (p < 0.001 based on a Welch Rank Sum test) on both platforms are indicated in black, on only the Ziplex platform are indicated in blue and on only the Affymetrix platform in green. B: Bland-Altman plots for expression values of all probes. Values determined to be outliers are indicated in the mean-difference (of the log2 fold change values) plot. A difference in log2 fold change of 0 is indicated by a solid black line. The upper and lower 95% limits of agreement for the difference in log2 fold change are indicated by red dashed lines, and arrows on the right hand side. Expression values that fall outside of these lines are considered outliers and are identified by their gene name.
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Figure 2: Comparison of the fold change difference in expression between NOSE and TOV samples for the Ziplex and Affymetrix platforms. A: The log2 fold change between the NOSE and TOV samples (mean NOSE signal intensity/mean TOV signal intensity) was calculated for the expression values of all 93 probes and plotted. Linear regression was performed resulting in the following model: log2 Affymetrix NOSE/TOV = 0.180098 + 1.0251794 log2 Ziplex NOSE/TOV with a Pearson's correlation coefficient (R) of 0.93. Probes that were not significant (p > 0.001 based on a Welch Rank Sum test) on either platform are indicated in grey, probes significant (p < 0.001 based on a Welch Rank Sum test) on both platforms are indicated in black, on only the Ziplex platform are indicated in blue and on only the Affymetrix platform in green. B: Bland-Altman plots for expression values of all probes. Values determined to be outliers are indicated in the mean-difference (of the log2 fold change values) plot. A difference in log2 fold change of 0 is indicated by a solid black line. The upper and lower 95% limits of agreement for the difference in log2 fold change are indicated by red dashed lines, and arrows on the right hand side. Expression values that fall outside of these lines are considered outliers and are identified by their gene name.

Mentions: As shown in Figure 2A, there was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between TOV versus NOSE samples (R = 0.93) and by Bland-Altman analysis (Figure 2B), where the majority of probes exhibited expression profiles in comparative analyses that fell within the 95% limits of agreement. Both statistical methods of comparative analysis of log2 fold differences show minimal variance as the mean increases regardless of the direction of expression difference evaluated: genes selected based on over- or under-expression in TOV samples relative to NOSE samples. Although there were examples of expression differences which fell outside the 95% limits of agreement as observed in the Bland-Altman analysis such as for RGSF4, PDPN, IGKC, IGHG1, C1QTNF1, TFF1 and IL1B (Figure 2B), both the directionality and magnitude of TOV versus NOSE expression patterns were generally consistent (Figure 2A and Table 2).


The chemiluminescence based Ziplex automated workstation focus array reproduces ovarian cancer Affymetrix GeneChip expression profiles.

Quinn MC, Wilson DJ, Young F, Dempsey AA, Arcand SL, Birch AH, Wojnarowicz PM, Provencher D, Mes-Masson AM, Englert D, Tonin PN - J Transl Med (2009)

Comparison of the fold change difference in expression between NOSE and TOV samples for the Ziplex and Affymetrix platforms. A: The log2 fold change between the NOSE and TOV samples (mean NOSE signal intensity/mean TOV signal intensity) was calculated for the expression values of all 93 probes and plotted. Linear regression was performed resulting in the following model: log2 Affymetrix NOSE/TOV = 0.180098 + 1.0251794 log2 Ziplex NOSE/TOV with a Pearson's correlation coefficient (R) of 0.93. Probes that were not significant (p > 0.001 based on a Welch Rank Sum test) on either platform are indicated in grey, probes significant (p < 0.001 based on a Welch Rank Sum test) on both platforms are indicated in black, on only the Ziplex platform are indicated in blue and on only the Affymetrix platform in green. B: Bland-Altman plots for expression values of all probes. Values determined to be outliers are indicated in the mean-difference (of the log2 fold change values) plot. A difference in log2 fold change of 0 is indicated by a solid black line. The upper and lower 95% limits of agreement for the difference in log2 fold change are indicated by red dashed lines, and arrows on the right hand side. Expression values that fall outside of these lines are considered outliers and are identified by their gene name.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Comparison of the fold change difference in expression between NOSE and TOV samples for the Ziplex and Affymetrix platforms. A: The log2 fold change between the NOSE and TOV samples (mean NOSE signal intensity/mean TOV signal intensity) was calculated for the expression values of all 93 probes and plotted. Linear regression was performed resulting in the following model: log2 Affymetrix NOSE/TOV = 0.180098 + 1.0251794 log2 Ziplex NOSE/TOV with a Pearson's correlation coefficient (R) of 0.93. Probes that were not significant (p > 0.001 based on a Welch Rank Sum test) on either platform are indicated in grey, probes significant (p < 0.001 based on a Welch Rank Sum test) on both platforms are indicated in black, on only the Ziplex platform are indicated in blue and on only the Affymetrix platform in green. B: Bland-Altman plots for expression values of all probes. Values determined to be outliers are indicated in the mean-difference (of the log2 fold change values) plot. A difference in log2 fold change of 0 is indicated by a solid black line. The upper and lower 95% limits of agreement for the difference in log2 fold change are indicated by red dashed lines, and arrows on the right hand side. Expression values that fall outside of these lines are considered outliers and are identified by their gene name.
Mentions: As shown in Figure 2A, there was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between TOV versus NOSE samples (R = 0.93) and by Bland-Altman analysis (Figure 2B), where the majority of probes exhibited expression profiles in comparative analyses that fell within the 95% limits of agreement. Both statistical methods of comparative analysis of log2 fold differences show minimal variance as the mean increases regardless of the direction of expression difference evaluated: genes selected based on over- or under-expression in TOV samples relative to NOSE samples. Although there were examples of expression differences which fell outside the 95% limits of agreement as observed in the Bland-Altman analysis such as for RGSF4, PDPN, IGKC, IGHG1, C1QTNF1, TFF1 and IL1B (Figure 2B), both the directionality and magnitude of TOV versus NOSE expression patterns were generally consistent (Figure 2A and Table 2).

Bottom Line: Overall, 75 of 93 (80.6%) genes exhibited consistent results in normal versus tumor tissue comparisons for both platforms (p < 0.001).There was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between tumor versus normal samples (R = 0.93) and by Bland-Altman analysis, where greater than 90% of expression values fell within the 95% limits of agreement.The reproducibility and ease-of-use of the technology suggests that the Ziplex array is a suitable platform for translational research.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Human Genetics, McGill University, Montreal, Canada. michael.quinn@mail.mcgill.ca

ABSTRACT

Background: As gene expression signatures may serve as biomarkers, there is a need to develop technologies based on mRNA expression patterns that are adaptable for translational research. Xceed Molecular has recently developed a Ziplex technology, that can assay for gene expression of a discrete number of genes as a focused array. The present study has evaluated the reproducibility of the Ziplex system as applied to ovarian cancer research of genes shown to exhibit distinct expression profiles initially assessed by Affymetrix GeneChip analyses.

Methods: The new chemiluminescence-based Ziplex gene expression array technology was evaluated for the expression of 93 genes selected based on their Affymetrix GeneChip profiles as applied to ovarian cancer research. Probe design was based on the Affymetrix target sequence that favors the 3' UTR of transcripts in order to maximize reproducibility across platforms. Gene expression analysis was performed using the Ziplex Automated Workstation. Statistical analyses were performed to evaluate reproducibility of both the magnitude of expression and differences between normal and tumor samples by correlation analyses, fold change differences and statistical significance testing.

Results: Expressions of 82 of 93 (88.2%) genes were highly correlated (p < 0.01) in a comparison of the two platforms. Overall, 75 of 93 (80.6%) genes exhibited consistent results in normal versus tumor tissue comparisons for both platforms (p < 0.001). The fold change differences were concordant for 87 of 93 (94%) genes, where there was agreement between the platforms regarding statistical significance for 71 (76%) of 87 genes. There was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between tumor versus normal samples (R = 0.93) and by Bland-Altman analysis, where greater than 90% of expression values fell within the 95% limits of agreement.

Conclusion: Overall concordance of gene expression patterns based on correlations, statistical significance between tumor and normal ovary data, and fold changes was consistent between the Ziplex and Affymetrix platforms. The reproducibility and ease-of-use of the technology suggests that the Ziplex array is a suitable platform for translational research.

Show MeSH
Related in: MedlinePlus