title: Quantification of the levels of CYP2D6 mRNA in peripheral blood leukocytes by reverse transcriptase polymerase chain reaction HPLC.
abstract: We developed a sensitive high performance liquid chromatography (HPLC) method for determining CYP2D6 mRNA in peripheral blood leukocytes (PBL) by using competitive reverse transcriptase polymerase chain reaction (RT-PCR). The method is specific, reproducible, and sensitive enough to quantify the absolute amount of low and high abundant CYP2D6 mRNA. The native CYP2D6 transcript and the internal standard, a CYP2D6 deletion RNA, were amplified with similar efficiency in RT-PCR. The PCR products were separated as the corresponding peaks in optimized HPLC. The coefficients of variation for competitive RT-PCR and HPLC determination were 1.5-6.5% and 0.6-2.4%, respectively, showing high reproducibility and reliability. This approach could also be applicable to the quantification of mRNA expressing on various tissues, including PBL, of which the expression levels were so low that they were hard to determine by existing agarose gel electrophoresis methods.
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko, Sagamihara, Kanagawa 229-0195, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Nagano
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hosono
authors.forename: Fumiko
authors.initials: F
authors.lastname: Negishi
authors.forename: Mashiho
authors.initials: M
authors.lastname: Yanagi
authors.forename: Masato
authors.initials: M
authors.lastname: Homma
authors.forename: Yukinao
authors.initials: Y
authors.lastname: Kohda
authors.forename: Machiko
authors.initials: M
authors.lastname: Watanabe
mesh_terms: D002851:Chromatography, High Pressure Liquid; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000097:blood* / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D012333:RNA, Messenger; D019389:Cytochrome P-450 CYP2D6