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Title of Journal: Eur J Clin Pharmacol

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Abbravation: European Journal of Clinical Pharmacology

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Springer-Verlag

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10.1016/0008-8749(78)90083-7

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1432-1041

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Real-time RT-PCR methodology for quantification of thiopurine methyltransferase gene expression

Authors: Malin Lindqvist, Sven Almer, Curt Peterson, Peter Söderkvist,

Publish Date: 2003/06/19
Volume: 59, Issue:3, Pages: 207-211
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Abstract

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.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)-methionine 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.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.This study was supported by grants from the Swedish Cancer Society, the Swedish Children Cancer Foundation, the Jenny Nordqvist Foundation and the Health research council in the south-east of Sweden (FORSS 2000–312, 2002–305). The authors thank Britt Sigfridsson for her excellent technical assistance and Isaac Austin for linguistic session. The experiments described here comply with the current laws of Sweden.


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