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Real-time RT-PCR me...
Real-time RT-PCR methodology for quantification of thiopurine methyltransferase gene expression
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- Lindqvist Appell, Malin, 1976- (författare)
- Linköpings universitet,Klinisk farmakologi,Hälsouniversitetet
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- Almer, Sven, 1953- (författare)
- Östergötlands Läns Landsting,Linköpings universitet,Hälsouniversitetet,Gastroenterologi och hepatologi,EMT-magtarm
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- Peterson, Curt, 1944- (författare)
- Östergötlands Läns Landsting,Linköpings universitet,Hälsouniversitetet,Klinisk farmakologi
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- Söderkvist, Peter, 1953- (författare)
- Linköpings universitet,Cellbiologi,Hälsouniversitetet
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(creator_code:org_t)
- Springer Science and Business Media LLC, 2003
- 2003
- Engelska.
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Ingår i: European Journal of Clinical Pharmacology. - : Springer Science and Business Media LLC. - 0031-6970 .- 1432-1041. ; 59:3, s. 207-211
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
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- 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.
Nyckelord
- MEDICINE
- MEDICIN
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