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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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DOI

10.1016/0008-8749(78)90083-7

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1432-1041

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Realtime RTPCR methodology for quantification of

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 realtime reversetranscription polymerase chain reaction RTPCR 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 cellsTPMT 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 614Cmethylmercaptopurine from 6MP using SadenosylL14Cmethylmethionine as methyl donor Thirtynine individuals were included in the study A cutoff value of 9 U/ml pRBC was used to distinguish intermediate TPMT enzyme activity from high TPMT enzyme activitySequencing of the realtime RTPCR amplicon proved that the method was specific for the TPMT cDNA without coamplification of the highly similar TPMT processed pseudogene The intraassay coefficients of variation CVs as determined by the threshold cycle were 07 for TPMT and 05 for huCYC The interassay CVs were 15 for TPMT and 40 for huCYC The intra and interassay CVs as determined by the TPMT/huCYC ratio were 86 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=066 P=00001 However we did not find any statistically significant correlation in individuals with lower enzyme activity or when analysing the whole populationThis 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 southeast 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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