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Method Article
Rudolf Napieralski
Therawis Diagnostics GmbH, Munich, Germany
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
The therascreen PITX2 RGQ PCR assay is an optimized in vitro DNA methylation-specific quantitative real-time PCR test intended for the determination of the percent methylation ratio (PMR) in the pituitary homeobox 2 (PITX2) transcription factor gene promoter 2. The test uses bisulfite-converted genomic DNA derived from formalin-fixed paraffin-embedded tumor tissues of breast cancer patients. The PMR will aid clinicians in selecting adjuvant systemic chemotherapy, e.g. anthracycline-based chemotherapy in high-risk lymph node-positive, estrogen receptor-positive, HER2-negative breast cancer patients. The assay is intended to be used by qualified users, such as technicians, molecular biologists/clinical chemists, or physicians, trained in molecular biology techniques and in vitro diagnostic procedures. The complete workflow is streamlined for medium sample throughput with highly reliable and robust readout and can be performed in two working days.
Keywords
PITX2, Breast Cancer, predictive, tumor biomarker, cancer, Epigenetics
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The authors declare the following potential conflict of interest: OW, GS, RN, ES and MS (†) are employees of Therawis Diagnostics. JL and JP are employees of QIAGEN. E.P., O.M., A.B., S.S. are employees of Halio Dx.
OW, RN, MK, VM and MS are equity owner of Therawis Diagnostics. The work was in part performed under a research agreement supported by Therawis Diagnostics.
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.pdf
Table 9 Delta PMR ranges for quality control of intra assay variation.
- supplement0.pdf
Table 7 Pipetting scheme of qPCR Mastermix and sample distribution.
- supplement0.pdf
Table 4 List of recommended equipment necessary for processing the overall workflow.
- supplement0.pdf
Table 5 Set-up of bisulfite conversion mix
- supplement0.pdf
Table 6 Bisulfite conversion thermal cycler conditions.
- supplement0.pdf
Table 3 List of potential hazardous reagents used in the workflow and their respective safety actions.
- supplement0.pdf
Table 2 List of necessary reagents and consumables
- supplement0.pdf
Table 1 Protocol steps and requirements
- supplement0.pdf
Table 10 qPCR validity criteria
- supplement0.pdf
Table 8 Troubleshooting guide
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An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Rudolf Napieralski
Therawis Diagnostics GmbH, Munich, Germany
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 20 Feb, 2018
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Genetics
License:
This work is licensed under a CC BY 4.0 License. Read Full License
The therascreen PITX2 RGQ PCR assay is an optimized in vitro DNA methylation-specific quantitative real-time PCR test intended for the determination of the percent methylation ratio (PMR) in the pituitary homeobox 2 (PITX2) transcription factor gene promoter 2. The test uses bisulfite-converted genomic DNA derived from formalin-fixed paraffin-embedded tumor tissues of breast cancer patients. The PMR will aid clinicians in selecting adjuvant systemic chemotherapy, e.g. anthracycline-based chemotherapy in high-risk lymph node-positive, estrogen receptor-positive, HER2-negative breast cancer patients. The assay is intended to be used by qualified users, such as technicians, molecular biologists/clinical chemists, or physicians, trained in molecular biology techniques and in vitro diagnostic procedures. The complete workflow is streamlined for medium sample throughput with highly reliable and robust readout and can be performed in two working days.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Figure 13
Figure 14
The authors declare the following potential conflict of interest: OW, GS, RN, ES and MS (†) are employees of Therawis Diagnostics. JL and JP are employees of QIAGEN. E.P., O.M., A.B., S.S. are employees of Halio Dx.
OW, RN, MK, VM and MS are equity owner of Therawis Diagnostics. The work was in part performed under a research agreement supported by Therawis Diagnostics.
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.pdf
Table 9 Delta PMR ranges for quality control of intra assay variation.
- supplement0.pdf
Table 7 Pipetting scheme of qPCR Mastermix and sample distribution.
- supplement0.pdf
Table 4 List of recommended equipment necessary for processing the overall workflow.
- supplement0.pdf
Table 5 Set-up of bisulfite conversion mix
- supplement0.pdf
Table 6 Bisulfite conversion thermal cycler conditions.
- supplement0.pdf
Table 3 List of potential hazardous reagents used in the workflow and their respective safety actions.
- supplement0.pdf
Table 2 List of necessary reagents and consumables
- supplement0.pdf
Table 1 Protocol steps and requirements
- supplement0.pdf
Table 10 qPCR validity criteria
- supplement0.pdf
Table 8 Troubleshooting guide
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