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Real-time RT-PCR methodology for quantification of thiopurine methyltransferase gene expression
Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0002-2809-7591
Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, MC - Medicincentrum, EMT-magtarm.
Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
2003 (English)In: European Journal of Clinical Pharmacology, ISSN 0031-6970, E-ISSN 1432-1041, Vol. 59, no 3, 207-211 p.Article in journal (Refereed) Published
Abstract [en]

Objective: The aim of the present study was to develop a real-time reverse-transcription polymerase chain reaction (RT-PCR) methodology for the quantification of thiopurine methyltransferase (TPMT) gene expression in whole blood and compare it with the TPMT enzyme activity measured in red blood cells. Methods: TPMT gene expression was quantified relative to the housekeeping gene cyclophilin (huCYC) and expressed as a TPMT/huCYC ratio. TPMT activity in red blood cells was determined by measuring the formation rate of 6- 14C-methylmercaptopurine from 6-MP using S-adenosyl-L-( 14C-methyl)-methlonine as methyl donor. Thirty-nine individuals were included in the study. A cut-off value of 9 U/ml pRBC was used to distinguish intermediate TPMT enzyme activity from high TPMT enzyme activity. Results: Sequencing of the real-time RT-PCR amplicon proved that the method was specific for the TPMT cDNA, without co-amplification of the highly similar TPMT processed pseudogene. The intra-assay coefficients of variation (CVs), as determined by the threshold cycle, were 0.7% for TPMT and 0.5% for huCYC. The interassay CVs were 1.5% for TPMT and 4.0% for huCYC. The intra- and interassay CVs, as determined by the TPMT/huCYC ratio, were 8.6% and 25%, respectively. There was a statistically significant correlation between TPMT enzyme activity and mRNA level in blood cells from individuals with an enzyme activity above 9 U/ml pRBC (rs = 0.66, P = 0.0001). However, we did not find any statistically significant correlation in individuals with lower enzyme activity or when analysing the whole population. Conclusion: We present a specific and robust real-time RT-PCR method for quantifying TPMT gene expression. The method may be used for studies on TPMT gene regulation.

Place, publisher, year, edition, pages
2003. Vol. 59, no 3, 207-211 p.
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Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-25409DOI: 10.1007/s00228-003-0617-zLocal ID: 9853OAI: oai:DiVA.org:liu-25409DiVA: diva2:245738
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13

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Lindqvist Appell, MalinAlmer, SvenPeterson, CurtSöderkvist, Peter

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Clinical PharmacologyFaculty of Health SciencesGastroenterology and HepatologyEMT-magtarmDepartment of Clinical PharmacologyCell biology
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European Journal of Clinical Pharmacology
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