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Biomarker discovery for colon cancer using a 761 gene RT-PCR assay.

Clark-Langone KM, Wu JY, Sangli C, Chen A, Snable JL, Nguyen A, Hackett JR, Baker J, Yothers G, Kim C, Cronin MT - BMC Genomics (2007)

Bottom Line: Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays.PCR was performed as independent TaqMan reactions for each candidate gene.We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity, sensitivity and reproducibility that are characteristics of RT-PCR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genomic Health, Inc, Redwood City, CA, USA. klangone@genomichealth.com

ABSTRACT

Background: Reverse transcription PCR (RT-PCR) is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE) clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis) and the likelihood of tumor response to standard chemotherapy regimens (prediction). We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application.

Results: RNA was extracted from formalin fixed paraffin embedded (FPE) tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery.

Conclusion: We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity, sensitivity and reproducibility that are characteristics of RT-PCR. Biomarkers discovered using this approach can be transferred to a clinical reference laboratory setting without having to re-validate the assay on a second technology platform.

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Related in: MedlinePlus

Comparison of paired whole plate average CT values for all patients. Expression analysis for 761 unique genes required each patient RNA sample to be divided between two 384 well plates. Shown here is the average raw CT value for all wells of data obtained for plate one plotted against the average raw CT obtained for plate two, for each patient. The patient sample which appears to be an outlier was re-assayed through RT-PCR and on repeat analysis, fell into alignment with the other samples. The solid line represents the least squares line fit and the dashed line represents the line of concordance.
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Figure 6: Comparison of paired whole plate average CT values for all patients. Expression analysis for 761 unique genes required each patient RNA sample to be divided between two 384 well plates. Shown here is the average raw CT value for all wells of data obtained for plate one plotted against the average raw CT obtained for plate two, for each patient. The patient sample which appears to be an outlier was re-assayed through RT-PCR and on repeat analysis, fell into alignment with the other samples. The solid line represents the least squares line fit and the dashed line represents the line of concordance.

Mentions: Two 384 well plates were prepared per patient RNA specimen to screen all 761 candidate marker genes. Assays were randomly assigned between the two plates except for reference genes which were present on both assay plates. The average CT values for each plate pair were compared and the data are illustrated in Figure 6. This graph demonstrates that plates from the same patient give consistent raw mean CT values prior to reference normalization. The one outlier specimen was repeated, and on repeat fell into alignment with the other plate results. It should be noted that Figure 6 represents all patient samples taken through RT-PCR, including the 23 samples run with less than 1 ng/well input RNA. These specimens, which had high average plate CT values were excluded from the final study analysis due to unsatisfactory RT-PCR data.


Biomarker discovery for colon cancer using a 761 gene RT-PCR assay.

Clark-Langone KM, Wu JY, Sangli C, Chen A, Snable JL, Nguyen A, Hackett JR, Baker J, Yothers G, Kim C, Cronin MT - BMC Genomics (2007)

Comparison of paired whole plate average CT values for all patients. Expression analysis for 761 unique genes required each patient RNA sample to be divided between two 384 well plates. Shown here is the average raw CT value for all wells of data obtained for plate one plotted against the average raw CT obtained for plate two, for each patient. The patient sample which appears to be an outlier was re-assayed through RT-PCR and on repeat analysis, fell into alignment with the other samples. The solid line represents the least squares line fit and the dashed line represents the line of concordance.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1994687&req=5

Figure 6: Comparison of paired whole plate average CT values for all patients. Expression analysis for 761 unique genes required each patient RNA sample to be divided between two 384 well plates. Shown here is the average raw CT value for all wells of data obtained for plate one plotted against the average raw CT obtained for plate two, for each patient. The patient sample which appears to be an outlier was re-assayed through RT-PCR and on repeat analysis, fell into alignment with the other samples. The solid line represents the least squares line fit and the dashed line represents the line of concordance.
Mentions: Two 384 well plates were prepared per patient RNA specimen to screen all 761 candidate marker genes. Assays were randomly assigned between the two plates except for reference genes which were present on both assay plates. The average CT values for each plate pair were compared and the data are illustrated in Figure 6. This graph demonstrates that plates from the same patient give consistent raw mean CT values prior to reference normalization. The one outlier specimen was repeated, and on repeat fell into alignment with the other plate results. It should be noted that Figure 6 represents all patient samples taken through RT-PCR, including the 23 samples run with less than 1 ng/well input RNA. These specimens, which had high average plate CT values were excluded from the final study analysis due to unsatisfactory RT-PCR data.

Bottom Line: Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays.PCR was performed as independent TaqMan reactions for each candidate gene.We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity, sensitivity and reproducibility that are characteristics of RT-PCR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genomic Health, Inc, Redwood City, CA, USA. klangone@genomichealth.com

ABSTRACT

Background: Reverse transcription PCR (RT-PCR) is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE) clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis) and the likelihood of tumor response to standard chemotherapy regimens (prediction). We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application.

Results: RNA was extracted from formalin fixed paraffin embedded (FPE) tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery.

Conclusion: We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity, sensitivity and reproducibility that are characteristics of RT-PCR. Biomarkers discovered using this approach can be transferred to a clinical reference laboratory setting without having to re-validate the assay on a second technology platform.

Show MeSH
Related in: MedlinePlus