| 1. |
- Farnebäck, Malin, 1972-
(författare)
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Quantitative analysis of melanoma transcripts : with emphasis on methodological and biological variation
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The introduction of RT-PCR made it possible to detect circulating tumor cells in melanoma patients by analysis of melanoma-associated transcripts, especially tyrosinase. Since the development of the first PCR method for tyrosinase mRNA, several studies have presented varying results. In the present thesis I have developed and used quantitative PCR methods in order to evaluate methodological and biological factors which may explain the disparity in the literature.Two methods were developed for tyrosinase, one competitive PCR and one real-time PCR method. With the real-time PCR technique, quantitative methods were also developed for tyrosinase related protein (TRP)-1, TRP-2, MART-1/Melan-A, S-100, GD2 synthase, MAGE-A3 and MAGE-A12. Methodological studies on the RNA extraction showed that the silica based RNA extraction method QIAamp gave considerably higher yields compared with the phenol-chloroform based extraction methods Ultraspec and FastRNA. Further studies showed that yields comparable with the QIAamp method could be obtained with the mRNA extraction method GenoPrep. Optimization of the cDNA synthesis procedure revealed that the reverse transcriptase and factors in the RNA sample inhibited the following PCR. This was avoided by diluting the cDNA sample before PCR.The stability of the tumor cell RNA in the samples is of great concern when it comes to transporting samples from distant hospitals to the laboratory. It was found that blood collected in ACD was best, although insufficiently, stabilized when stored at +4 °C compared with room temperature. Similar stability was also obtained for PAXgene tubes stored at room temperature, however the stability of RNA was much improved when the PAXgene tubes were frozen.Studies on the biological variation in cultured melanoma cell lines and tissue sections from melanoma metastases showed that the expression of melanoma associated transcripts varied widely. In melanoma cell lines the expression of the transcripts tyrosinase, TRP-1, TRP-2 and MART-1/Melan-A was related to the pigmentation of the cell lines. The pigment-related transcripts and S-100 were expressed at higher levels than GD2 synthase, MAGE-A3 and MAGE-A12 in the melanoma metastases. The expressions of TRP-2 and GD2 synthase appeared to be influenced by therapy. In metastases from patients treated with a combination of cisplatinum, DTIC and interferon-α2b, TRP-2 was expressed at higher levels and GD2 synthase at lower levels compared with untreated patients.
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| 2. |
- Kågedal, Bertil, 1943-, et al.
(författare)
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How useful are housekeeping genes? Variable expression in melanoma metastases
- 2007
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Ingår i: Clinical Chemistry and Laboratory Medicine. - 1434-6621 .- 1437-4331. ; 45:11, s. 1481-1487
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is a certain difference in opinion regarding the optimal choice of housekeeping genes used as normalization factors in gene expression analysis. We have therefore examined the suitability of three housekeeping genes, hypoxanthine phosphoribosyl transferase, beta(2)-glucuronidase and beta(2)-microglobulin, for normalization of expression data from melanoma metastases. Methods: The expression of the three housekeeping genes was quantified by quantitative reverse transcription PCR in snap-frozen sections from 44 melanoma metastases, of which 19 were from patients treated with cisplatinum, dacarbazine and interferon alpha 2b. Results: The expression of each housekeeping gene varied considerably between the different metastases. Histopathological examination of the tissue sections revealed variation in the amount of tumor cells in the tissue, necrosis, varying degrees of lymphocyte infiltration, and lymph node remnants. Based on this examination, 16 biopsies were omitted from further analysis because they had cracked, contained empty or necrotic areas, or were dominated by lymph node tissue. Even in sections with more than 90% tumor cells, a wide variation in the expression of the three housekeeping genes was found. The amount of lymphatic infiltrate in the tumors can have an effect on the expression of housekeeping genes in the metastases, whereas treatment did not seem to influence the expression. Conclusions: We conclude that the choice of housekeeping genes can have great impact on the normalization of specific genes in melanoma metastases. Furthermore, in the analysis of mRNA expression in tumor tissue, microscopic examination is of great importance to evaluate the integrity and cellular composition of the biopsy.
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| 3. |
- Kågedal, Bertil, 1943-, et al.
(författare)
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Pterin-dependent tyrosine hydroxylase mRNA is not expressed in human melanocytes or melanoma cells
- 2004
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Ingår i: Pigment Cell Research. - : Wiley. - 0893-5785 .- 1600-0749. ; 17:4, s. 346-351
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Tidskriftsartikel (refereegranskat)abstract
- Pterin-dependent tyrosine hydroxylase has been described to occur occasionally in melanocytes. It is therefore important to quantify the mRNA of this enzyme in pigment cells to understand whether this enzyme can take an active part in pigment formation. A real-time reverse transcription-polymerase chain reaction method was used to quantify tyrosine hydroxylase mRNA in melanocytes and melanoma cells. The calibrator was obtained by amplification of a segment of cDNA from tyrosine hydroxylase mRNA, which included the target thus allowing enumeration of the number of transcripts per cell. In melanocytes (n = 3), tyrosine hydroxylase mRNA ranged from non-detectable to 0.000492 transcripts/cell and in melanoma cells from non-detectable to 0.005340 transcripts/cell. In neuroblastoma cells, the median tyrosine hydroxylase mRNA number was 0.4 transcripts/cell (range 0.02-25 transcripts/cell). The amount of tyrosine hydroxylase mRNA in the pigment cells was far less than the mRNA concentrations of four melanocyte-specific proteins measured in the same melanocytes and melanoma cells. We conclude that on the average less than 1 of 1000 melanocytes and melanoma cells contains at least one tyrosine hydroxylase mRNA molecule. Consequently, in 999 of 1000 cells translation into the corresponding enzyme protein cannot occur because of the lack of an mRNA template. Thus, in these cells there is no pterin-dependent tyrosine hydroxylase that can contribute to pigment formation by producing priming amounts of L-dopa for proper function of tyrosinase.
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