Reproducibility of detection of tyrosinase and MART-1 transcripts in the peripheral blood of melanoma patients: a quality control study using real-time quantitative RT-PCR
| Autor: | S.C. van den Bosch, Th. Wobbes, Dirk J. Ruiter, Ewald J.B.M. Mensink, G.N.P. van Muijen, T.J. de Vries, Cornelis J. A. Punt, M.J.M. de Rooij, A.T.F. van de Locht, A. Fourkour |
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| Přispěvatelé: | Other departments |
| Jazyk: | angličtina |
| Rok vydání: | 1999 |
| Předmět: |
Male
Cancer Research Transcription Genetic MelanA/MART-1 Tyrosinase causes and effects (sepsis and inflammation) [Sepsis and non-bacterial generalized inflammation] law.invention law MART-1 Antigen Innovation of diagnosis and therapy in clinical dermatology quantitative RT-PCR Polymerase chain reaction Aged 80 and over Monophenol Monooxygenase Reverse Transcriptase Polymerase Chain Reaction Melanoma OVERIG ONDERZOEK MIES Regular Article Middle Aged circulating cancer cells Neoplastic Cells Circulating Hematopoiesis and stem cell transplantation Neoplasm Proteins Hydroxymethylbilane Synthase Reverse transcription polymerase chain reaction Real-time polymerase chain reaction Oncology Female Adult Quality Control Adolescent mogelijke oorzaken en gevolgen (sepsis en ontsteking) [Sepsis en niet-bacteriële gegeneraliseerde ontsteking] tyrosinase Biology Experimental diagnostics and therapy of malignancies Antigens Neoplasm Complementary DNA melanoma medicine Humans Tumor pathology neoplasms Aged Gene Amplification Reproducibility of Results Tumor pathologie medicine.disease Molecular biology Reverse transcriptase Immunology Innovatie van diagnostische en therapeutische mogelijkheden in de klinische dermatologie Tumorimmunology |
| Zdroj: | British Journal of Cancer, 80, 883-891 British Journal of Cancer, 80, pp. 883-891 British Journal of Cancer British journal of cancer, 80(5-6), 883-891. Nature Publishing Group |
| ISSN: | 0007-0920 |
| DOI: | 10.1038/sj.bjc.6690436 |
| Popis: | In recent years, large discrepancies were described in the success rate of the tyrosinase reverse transcription polymerase chain reaction (RT-PCR) for detecting melanoma cells in the peripheral blood of melanoma patients. We present a quality control study in which we analysed the reproducibility of detection of tyrosinase and MART-1 transcripts in 106 blood samples from 68 melanoma patients (mainly stages III and IV). With this study, we aimed to improve insight in the reproducibility of a RT-PCR for the detection of (minimal) amounts of circulating melanoma cells. We performed two reverse transcriptions on each mRNA sample and performed tyrosinase and MART-1 nested PCRs in duplicate per cDNA sample. Thus, four tyrosinase and four MART-1 measurements were performed per blood sample. In our study, the majority of blood samples was negative for tyrosinase (80%) or MART-1 (66%). Only four samples were positive in all four determinations for tyrosinase and seven for MART-1. Variable results (1–3 times positive results) were obtained for tyrosinase and MART-1 in 16% and 27% respectively. MART-1 PCR had a better performance than tyrosinase PCR. Sensitivity increased when both markers were used. We reasoned that the low number of melanoma marker PCR-positive blood samples can be explained by differences in mRNA quality. By using real-time quantitative PCR, we found that this was not the case: amplification of porphobilinogen deaminase (PBGD), a low copy household gene, was not different in blood samples in which a melanoma marker was not detected from groups in which this marker was detected more or less consistently (1–4 times). When applying real-time quantitative PCR for tyrosinase and MART-1, we found that a low amount of SK-MEL-28 cell equivalents was present in the blood of melanoma patients, with a higher number of equivalents in the group with a consistently positive result. We conclude that low reproducibility of a repeated assay for the detection of circulating melanoma cells is not caused by differences in mRNA quality between the samples, but due to low numbers of amplifiable target mRNA molecules in the mRNA sample. Use of more than one marker and repetition of the assay will increase the probability of finding positive PCR results. © 1999 Cancer Research Campaign |
| Databáze: | OpenAIRE |
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