Quantification of the levels of CYP2D6 mRNA in peripheral blood leukocytes by reverse transcriptase polymerase chain reaction HPLC.
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.
Nagano
Satoko
S
Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko, Sagamihara, Kanagawa 229-0195, Japan.
Hosono
Hiroyuki
H
Negishi
Fumiko
F
Yanagi
Mashiho
M
Homma
Masato
M
Kohda
Yukinao
Y
Watanabe
Machiko
M
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
D012333:RNA, Messenger; D019389:Cytochrome P-450 CYP2D6