title: Real-time RT-PCR methodology for quantification of thiopurine methyltransferase gene expression.
abstract: 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-(14)C-methylmercaptopurine from 6-MP using S-adenosyl-L-((14)C-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.
  
  
  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 (r(s)=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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medicine and Care, Division of Clinical Pharmacology, Faculty of Health Sciences, Linköping University, 58185 Linköping, Sweden. malin.lindqvist@imv.liu.se
authors.forename: Malin
authors.initials: M
authors.lastname: Lindqvist
authors.forename: Sven
authors.initials: S
authors.lastname: Almer
authors.forename: Curt
authors.initials: C
authors.lastname: Peterson
authors.forename: Peter
authors.initials: P
authors.lastname: Söderkvist
mesh_terms: D004912:Erythrocytes / Q000737:chemistry / Q000201:enzymology*; D015870:Gene Expression; D006801:Humans; D015122:Mercaptopurine / Q000031:analogs & derivatives* / Q000097:blood; D008780:Methyltransferases / Q000096:biosynthesis / Q000097:blood* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
chemical_list: D012333:RNA, Messenger; C010240:6-methylthiopurine; D015122:Mercaptopurine; D008780:Methyltransferases; C022745:thiopurine methyltransferase