Quantitative RT-PCR on CYP1A1 heterogeneous nuclear RNA: a surrogate for the in vitro transcription run-on assay.
A quantitative reverse transcription polymerase chain reaction (RT-PCR) assay was developed to amplify a region of the CYP1A1 heterogeneous nuclear RNA (hnRNA) transcript encompassing the first intron-exon boundary. The RT-PCR protocol uses a CYP1A1 recombinant RNA internal standard identical to the target hnRNA except for an engineered unique internal restriction site. Its inclusion enables normalization between reactions and a measurement of the absolute number of target hnRNA transcripts. Specificity for the hnRNA was achieved by using intron-directed primers in both the RT and the PCR. Nuclear run-on assays and the hnRNA RT-PCR assay detected an equivalent increase in transcription of Cyp1a-1 in cultured murine Hepa 1c1c7 cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The RT-PCR assay also revealed TCDD-dependent transcriptional activation of the Cyp1a-1 gene in murine skin, a tissue unsuited to the nuclear run-on assay because of inherent difficulties associated with the isolation of nuclei. These examples demonstrate that the hnRNA RT-PCR assay is a facile surrogate for the nuclear run-on assay. Moreover, the sensitivity and design characteristics of the RT-PCR assay suggest the potential for its broad application in general transcriptional research.
Elferink
C J
CJ
Institute of Chemical Toxicology, Wayne State University, Detroit, MI 48201, USA. celferi@cms.cc.wayne.edu
Reiners
J J
JJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific; D004790:Enzyme Induction / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D015603:Keratinocytes; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012313:RNA / Q000235:genetics / Q000592:standards*; D012332:RNA, Heterogeneous Nuclear / Q000032:analysis* / Q000235:genetics; D012015:Reference Standards; D014158:Transcription, Genetic
D000072317:Polychlorinated Dibenzodioxins; D012332:RNA, Heterogeneous Nuclear; C039919:RNA, recombinant; D012313:RNA; D003577:Cytochrome P-450 Enzyme System; C052380:endodeoxyribonuclease NcoI; D015252:Deoxyribonucleases, Type II Site-Specific