title: An RT-PCR-based strategy to estimate full-length CYP2D6 mRNA copy number.
abstract: 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.
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, Washington 98195-7610, USA.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: McConnachie
authors.forename: Miklos
authors.initials: M
authors.lastname: Bodor
authors.forename: Brian
authors.initials: B
authors.lastname: Phillips
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Kelly
authors.forename: Danny D
authors.initials: DD
authors.lastname: Shen
authors.forename: Rodney J Y
authors.initials: RJ
authors.lastname: Ho
mesh_terms: 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*
chemical_list: D012322:RNA Precursors; D012333:RNA, Messenger; D019389:Cytochrome P-450 CYP2D6