Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cells
<p>Abstract</p> <p>Background</p> <p>Immunomagnetic enrichment followed by RT-PCR (immunobead RT-PCR) is an efficient methodology to identify disseminated carcinoma cells in the blood and bone marrow. The RT-PCR assays must be both specific for the tumor cells and suffi...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2002-05-01
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| Series: | BMC Cancer |
| Online Access: | http://www.biomedcentral.com/1471-2407/2/14 |
| _version_ | 1818065932009865216 |
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| author | Walsh David CA Stephenson Sally-Anne Raynor Michael Pittman Kenneth B Dobrovic Alexander |
| author_facet | Walsh David CA Stephenson Sally-Anne Raynor Michael Pittman Kenneth B Dobrovic Alexander |
| author_sort | Walsh David CA |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Immunomagnetic enrichment followed by RT-PCR (immunobead RT-PCR) is an efficient methodology to identify disseminated carcinoma cells in the blood and bone marrow. The RT-PCR assays must be both specific for the tumor cells and sufficiently sensitive to enable detection of single tumor cells. We have developed a method to test RT-PCR assays for any cancer. This has been investigated using a panel of RT-PCR markers suitable for the detection of breast cancer cells.</p> <p>Methods</p> <p>In the assay, a single cell line-derived tumor cell is added to 100 peripheral blood mononuclear cells (PBMNCs) after which mRNA is isolated and reverse transcribed for RT-PCR analysis. PBMNCs without added tumor cells are used as specificity controls. The previously studied markers epidermal growth factor receptor (<it>EGFR</it>), mammaglobin 1 (<it>MGB1</it>), epithelial cell adhesion molecule (<it>EpCAM</it>/<it>TACSTD1</it>), mucin 1 (<it>MUC1</it>), carcinoembryonic antigen (<it>CEA</it>) were tested. Two new epithelial-specific markers <it>ELF3</it> and <it>EphB4</it> were also tested.</p> <p>Results</p> <p><it>MUC1</it> was unsuitable as strong amplification was detected in 100 cell PBMNC controls. Expression of <it>ELF3</it>, <it>EphB4</it>, <it>EpCAM</it>, <it>EGFR</it>, <it>CEA</it> and <it>MGB1</it> was found to be both specific for the tumor cell, as demonstrated by the absence of a signal in most 100 cell PBMNC controls, and sensitive enough to detect a single tumor cell in 100 PBMNCs using a single round of RT-PCR.</p> <p>Conclusions</p> <p><it>ELF3</it>, <it>EphB4</it>, <it>EpCAM</it>, <it>EGFR</it>, <it>CEA</it> and <it>MGB1</it> are appropriate RT-PCR markers for use in a marker panel to detect disseminated breast cancer cells after immunomagnetic enrichment.</p> |
| first_indexed | 2024-12-10T14:59:44Z |
| format | Article |
| id | doaj.art-8df3d617922f4b3e90c01843f86749d1 |
| institution | Directory Open Access Journal |
| issn | 1471-2407 |
| language | English |
| last_indexed | 2024-12-10T14:59:44Z |
| publishDate | 2002-05-01 |
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| series | BMC Cancer |
| spelling | doaj.art-8df3d617922f4b3e90c01843f86749d12022-12-22T01:44:13ZengBMCBMC Cancer1471-24072002-05-01211410.1186/1471-2407-2-14Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cellsWalsh David CAStephenson Sally-AnneRaynor MichaelPittman Kenneth BDobrovic Alexander<p>Abstract</p> <p>Background</p> <p>Immunomagnetic enrichment followed by RT-PCR (immunobead RT-PCR) is an efficient methodology to identify disseminated carcinoma cells in the blood and bone marrow. The RT-PCR assays must be both specific for the tumor cells and sufficiently sensitive to enable detection of single tumor cells. We have developed a method to test RT-PCR assays for any cancer. This has been investigated using a panel of RT-PCR markers suitable for the detection of breast cancer cells.</p> <p>Methods</p> <p>In the assay, a single cell line-derived tumor cell is added to 100 peripheral blood mononuclear cells (PBMNCs) after which mRNA is isolated and reverse transcribed for RT-PCR analysis. PBMNCs without added tumor cells are used as specificity controls. The previously studied markers epidermal growth factor receptor (<it>EGFR</it>), mammaglobin 1 (<it>MGB1</it>), epithelial cell adhesion molecule (<it>EpCAM</it>/<it>TACSTD1</it>), mucin 1 (<it>MUC1</it>), carcinoembryonic antigen (<it>CEA</it>) were tested. Two new epithelial-specific markers <it>ELF3</it> and <it>EphB4</it> were also tested.</p> <p>Results</p> <p><it>MUC1</it> was unsuitable as strong amplification was detected in 100 cell PBMNC controls. Expression of <it>ELF3</it>, <it>EphB4</it>, <it>EpCAM</it>, <it>EGFR</it>, <it>CEA</it> and <it>MGB1</it> was found to be both specific for the tumor cell, as demonstrated by the absence of a signal in most 100 cell PBMNC controls, and sensitive enough to detect a single tumor cell in 100 PBMNCs using a single round of RT-PCR.</p> <p>Conclusions</p> <p><it>ELF3</it>, <it>EphB4</it>, <it>EpCAM</it>, <it>EGFR</it>, <it>CEA</it> and <it>MGB1</it> are appropriate RT-PCR markers for use in a marker panel to detect disseminated breast cancer cells after immunomagnetic enrichment.</p>http://www.biomedcentral.com/1471-2407/2/14 |
| spellingShingle | Walsh David CA Stephenson Sally-Anne Raynor Michael Pittman Kenneth B Dobrovic Alexander Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cells BMC Cancer |
| title | Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cells |
| title_full | Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cells |
| title_fullStr | Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cells |
| title_full_unstemmed | Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cells |
| title_short | Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cells |
| title_sort | optimisation of the rt pcr detection of immunomagnetically enriched carcinoma cells |
| url | http://www.biomedcentral.com/1471-2407/2/14 |
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