An RT-PCR-based strategy to estimate full-length CYP2D6 mRNA copy number.
The goal of this study is to develop an analytical tool to assess cytochrome P450 2D6 (CYP2D6) levels in the form of full-length transcripts. CYP2D6 RNA in test samples was evaluated by co-amplification with an internal RNA control in a reverse-transcribed polymerase chain reaction (RT-PCR). The internal CYP2D6 RNA control was constructed by internally deleting 474 bp of CYP2D6 RNA, allowing simultaneous amplification of the test RNA together with the internal control RNA in a single RT-PCR reaction. With sequential dilution of test RNA, the CYP2D6 mRNA transcript levels in test samples were estimated. The full-length RT-PCR strategy allowed semiquantitative assessments of CYP2D6 RNA transcripts with a sensitivity limit of 500 copies for CYP2D6 RNA transcripts, 2500 copies/microg total human liver RNA, and 10% intraday coefficient of variation (CV). In a method validation study, the CYP2D6 activity appeared to relate more closely to full-length CYP2D6 mRNA concentration than a short-sequence of CYP2D6 RNA estimated with a real-time quantitative RT-PCR assay. We have developed an efficient semiquantitative assay and demonstrated its suitability for estimating full-length CYP2D6 mRNA transcripts in cells and tissues.
McConnachie
Lisa A
LA
Department of Pharmaceutics, University of Washington, Seattle, Washington 98195-7610, USA.
Bodor
Miklos
M
Phillips
Brian
B
Kelly
Edward J
EJ
Shen
Danny D
DD
Ho
Rodney J Y
RJ
D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D006801:Humans; D012322:RNA Precursors / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012322:RNA Precursors; D012333:RNA, Messenger; D019389:Cytochrome P-450 CYP2D6