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Stability of blood samples for analysis of tumour cell transcripts
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.
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

When mRNA expression from tumor cells is analyzed in blood, one of the greatest problems is to keep the samples stable during transfer to the laboratory. Improved systems for stabilization of transcripts therefore have to be developed.

Blood was collected in PAXgene™ Blood RNA Tubes and spiked with melanoma and neuroblastoma cells. The stability of tumor transcripts at room temperature was tested after 1, 2, 3, 5, 7, and 9 days. PAXgene tubes were also frozen at -20 °C and -70 °C for 14 days. Total RNA was extracted by PAXgene™ Blood RNA Kit and mRNA was extracted by the GenoPrep Direct mRNA Kit. Tyrosinase, MAGE, tyrosine hydroxylase, and GD2 synthase mRNA was quantified by real-time PCR. The stability of these transcripts was compared with the stability in ACD tubes when RNA extraction was performed with QIAamp RNA Blood Mini Kit.

The yields of transcripts from the PAXgene tubes using the PAXgene™ Blood RNA Kit varied from 37% to 49% of the yields from ACD tubes. When mRNA was extracted with the GenoPrep Direct mRNA Kit the yields increased to 67-121%. Stability tests showed significant decreases of all the transcripts in the PAXgene tubes after two days at room temperature. After seven days, 10% of the initial transcript concentrations remained, whereas 32% remained in the ACD tubes. However, in the frozen PAXgene tubes the transcript levels were unchanged for 14 days.

We conclude that the PAXgene tube is suitable when the sample is stored at -20 °C or -70 °C and mRNA is extracted using GenoPrep Direct mRNA Kit.

Keyword [en]
RNA stability, real-time PCR, blood sampling, preanalytics, minimal residual disease
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-84993OAI: oai:DiVA.org:liu-84993DiVA: diva2:563477
Available from: 2012-10-30 Created: 2012-10-30 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.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 843
National Category
Medical and Health Sciences
Identifiers
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)
Opponent
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-30Bibliographically approved

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Farnebäck, MalinKullman, AnitaKågedal, Bertil

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