Quantification of the Levels of CYP2D6 mRNA in Peripheral Blood Leukocytes by Reverse Transcriptase Polymerase Chain Reaction HPLC

Abstract

Several methods have been reported for the semi-quantitative detection of small amounts of a particular mRNA in tissues through specific amplification of the extracted mRNA by reverse transcriptase polymerase chain reaction (RT-PCR), usually followed by agarose gel electrophoretic separation and ethidium bromide staining for fluorescence detection. [1][2][3][4] However, most of the methods are not suitable for accurate quantification owing to lack of correction for incomplete recovery of mRNA during extraction from the tissue or subsequent PCR procedures. It is also difficult to avoid PCR amplification into a non-linear region because of insufficient detection sensitivity. To overcome these problems, competitive RT-PCR with an internal standard has been shown to be a useful method for mRNA quantification, where high performance liquid chromatography (HPLC) with a UV detector was employed for detecting PCR products. 5-7)CYP2D6 with genetic polymorphism is involved in the oxidative metabolism of many drugs including neuroleptics, tricyclic antidepressants, selective serotonin reuptake inhibitors, b-adrenoreceptor blockers, and antiarrhythmics. 8,9) Although the genotyping of CYP2D6 is useful for assessing drug efficacy and adverse effects, it is hard to conduct routine clinical measurement because of the many single nucleotide polymorphisms (SNPs). Since blood is a readily accessible tissue, an appealing concept should be to use peripheral blood leukocytes (PBL) as a surrogate for CYP activities that manifest in internal organs. [10][11][12] In the present study, we developed a sensitive and quantitative RT-PCR-HPLC method for the measurement of CYP2D6 mRNA in PBL. ExperimentalPreparation of RNA Standards for RT-PCR wt 2D6, the standard cDNA containing 456 bp of CYP2D6 DNA (nt 1087-1542), was cloned to pBluescript KS (ϩ) vector by PCR from cDNA Library Human Liver (Takara Bio, Japan), and designated as pKSp2D6. For preparation of the internal standard, is 2D6, 68 bp (nt 1107-1153) was deleted from pKSp2D6 using PCR, and named as pKSd2D. The primers used for amplification of wt 2D6 were CGAGCAAGCTT-Ex7F, 5Ј-CGAGCAAGCTTGGAGATCG-ACGACGTGATAG-3Ј and AGGTCGTCGAC-Ex9R, 5Ј-AGGTCGTCGA-CACCAGGAAAGCAAAGACACC-3Ј. The primers used for amplification of is 2D6 were CGAGCAAGCTT-Ex7F D46, 5Ј-CGAGCAAGCTTGGA-GATCGACGACGTGATAGGTGCAGCGCTTTGGGGACGACAT-3Ј and AGGTCGTCGAC-Ex9R, 5Ј-AGGTCGTCGACACCAGGAAAGCAAA-GACACC-3Ј. The standard RNAs, wt 2D6 and is 2D6, were prepared using Riboprobe ® in vitro Transcription Systems (Promega, U.S.A.) from pKSp2D6 and pKSd2D6, respectively. The RNAs were treated with RNasefree DNase (QIAGEN, Netherlands) and purified by QIAamp ® RNA Blood Mini (QIAGEN). The RNA standards were diluted to the concentration of 1 pg/ml and stored in aliquots at Ϫ80°C until use.Subjects The study was explained to 8 healthy volunteers (2 male and 6 female) and written informed consent was obtained from each. The study was approved by the Institutional Review Board of the Faculty of Pharmaceutical Sciences, Teikyo University an...