Quantitative RT-PCR on CYP1A1 heterogeneous nuclear RNA: a surrogate for the in vitro transcription run-on assay

Biotechniques. 1996 Mar;20(3):470-7. doi: 10.2144/19962003470.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • Cell Nucleus / metabolism
  • Cytochrome P-450 Enzyme System / genetics*
  • Deoxyribonucleases, Type II Site-Specific
  • Enzyme Induction / drug effects
  • Genetic Vectors / genetics
  • Keratinocytes
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Polychlorinated Dibenzodioxins / pharmacology
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards
  • RNA / genetics
  • RNA / standards*
  • RNA, Heterogeneous Nuclear / analysis*
  • RNA, Heterogeneous Nuclear / genetics
  • Reference Standards
  • Transcription, Genetic*


  • Polychlorinated Dibenzodioxins
  • RNA, Heterogeneous Nuclear
  • RNA, recombinant
  • RNA
  • Cytochrome P-450 Enzyme System
  • endodeoxyribonuclease NcoI
  • Deoxyribonucleases, Type II Site-Specific