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How useful are housekeeping genes?: variable expression in melanoma metastases
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
Department of Oncology, University Hospital MAS and Lund University, Malmö, Sweden.
Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Cytology. Linköping University, Faculty of Health Sciences.
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2007 (English)In: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, Vol. 45, no 11, 1481-1487 p.Article in journal (Refereed) Published
Abstract [en]

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, β-glucuronidase and β2-micro-globulin, 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-α2b.

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 meta-stases, 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.

Place, publisher, year, edition, pages
2007. Vol. 45, no 11, 1481-1487 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-39472DOI: 10.1515/CCLM.2007.303Local ID: 48767OAI: diva2:260321
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2012-10-30
In thesis
1. Quantitative analysis of melanoma transcripts: with emphasis on methodological and biological variation
Open this publication in new window or tab >>Quantitative analysis of melanoma transcripts: with emphasis on methodological and biological variation
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2004. 61 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 843
National Category
Medical and Health Sciences
urn:nbn:se:liu:diva-24062 (URN)3621 (Local ID)91-7373-816-6 (ISBN)3621 (Archive number)3621 (OAI)
Public defence
2004-04-07, Aulan, Administrationshuset, Hälsouniversitetet, Linköping, 09:00 (Swedish)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-30Bibliographically approved

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Kågedal, BertilFarnebäck, MalinHåkansson, AnnikaGustafsson, Bertil
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