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Articles |
Departments of
1
Clinical Biochemistry and
2
Oncology, AKH, University Hospital in Aarhus, DK-8000 Aarhus, Denmark.
3
Department of Tumor Cell Biology, Institute of Cancer
Biology, Danish Cancer Society, DK-2100 Copenhagen, Denmark.
a Address correspondence to this author at: Department of Clinical Biochemistry, AKH, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Århus C, Denmark. Fax 45-89493060; e-mail
AKH.grp02s.bsl{at}aaa.dk
Background: Reverse transcription-PCR (RT-PCR) amplification of melanoma cell-specific mRNA can detect melanoma cells in the peripheral blood of patients with malignant melanoma. We present a method to quantify mRNA coding for the melanoma-specific melanoma antigen recognized by T cells #1 (MART-1) in RNA isolated from peripheral blood.
Methods: To establish a calibration curve, we measured the concentration of MART-1 mRNA in SK-MEL-28 melanoma cells grown in vitro by competitive RT-PCR. Serial dilutions of these cells were used as calibrators in the assay. The assay was conducted by adding a fixed amount of a RNA internal standard to RNA isolated from either peripheral blood or the calibrators before RT-PCR amplification with MART-1 primers in a nested PCR design. The amount of MART-1 mRNA in blood samples was calculated from the calibration curve.
Results: Addition of melanoma cells grown in vitro to blood from healthy donors demonstrated that the method can detect a single SK-MEL-28 melanoma cell in 1 mL of blood (1.5 x 10-21 mol MART-1 mRNA/mL). MART-1 mRNA was observed in 4 of 12 blood samples from patients with malignant melanoma, at concentrations of 3–18 x 10-21 mol MART-1 mRNA/mL of blood. No MART-1 mRNA was detected in blood samples from 25 controls without malignant melanoma. Intra- and interassay CVs were 15% (n = 12; mean = 44 x 10-21 mol MART-1 mRNA/mL) and 33% (15 samples analyzed in two different analytical runs; mean = 30 x 10-21 mol MART-1 mRNA/mL), respectively.
Conclusions: Our method is the first competitive RT-PCR assay for quantification of melanoma cells in blood samples that compensates for the variation of both the reverse transcription and PCR reactions. The method allows the inclusion of control samples for continuous quality assessment.
The following articles in journals at HighWire Press have cited this article:
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  H. N. Abrahamsen, E. Nexo, T. Steiniche, S. J. Hamilton-Dutoit, and B. S. Sorensen Quantification of Melanoma mRNA Markers in Sentinel Nodes: Pre-Clinical Evaluation of a Single-Step Real-Time Reverse Transcriptase-Polymerase Chain Reaction Assay J. Mol. Diagn., August 1, 2004; 6(3): 253 - 259. [Abstract] [Full Text]
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H. N. Abrahamsen, T. Steiniche, E. Nexo, S. J. Hamilton-Dutoit, and B. S. Sorensen Towards Quantitative mRNA Analysis in Paraffin-Embedded Tissues Using Real-Time Reverse Transcriptase-Polymerase Chain Reaction: A Methodological Study on Lymph Nodes from Melanoma Patients J. Mol. Diagn., February 1, 2003; 5(1): 34 - 41. [Abstract] [Full Text] [PDF]
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R. M. Kisabeth Laboratory Adaptations--Changing Expectations Clin. Chem., August 1, 2001; 47(8): 1509 - 1515. [Full Text] [PDF]
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