( mRNA was extracted with magneticol...)Sensitive detection of tumor cells in,,,peripheral blood of carcinoma patients,,,by a reverse transcription PCR method

mRNA was extracted with magnetic oligo-(dT) beads (DYNAL) followed by a high sensitive reverse transcription (QIAGEN).
AdnaTest CancerDetect
Expression analysis of tumor cells with multiplex PCR (up to five markers per assay) for the detection of mRNAs encoding for tumor markers. Multiplex PCR reactions were prepared using the cDNA from above as template, the primer mixture provided by AdnaGen and the HotStar PCR-kit (QIAGEN). PCR samples were analyzed using the Agilent 2100 bioanalyzer and the DNA 500 LabChip kit.
Inoculation system
Cell spiking experiments with different carcinoma cell lines for each kind of investigated cancer were used to test the potential lower detection limit of this technique. Defined numbers of 2-100 cells were added to 5-mL aliquots of whole blood from healthy donors, an unspiked blood sample was used as negative control, cancer cell lines served as positive controls. The specificity of the selected antibodies was determined by flow cytometry.
Results and discussion

AdnaGen developed a diagnostic system for early detection of disseminated cells in peripheral blood of testicular, breast and colorectal carcinoma patients. This diagnostic approach is designed to optimize enrichment and analysis of tumor cells. The specificity of the method is shown by immunofluorescence staining (figures 1 and 2) and the lower detection limit by RT-PCR (figure 3).
Specificity
Figure 1 shows the specific recognition of tumor cells by the selected antibodies coupled to magnetic beads (AdnaTest CancerSelect) by microscopy. The immunofluorescence assay (figure 2) documents the specificity of the antibody mixture (AdnaTest CancerSelect) used
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