BioTechniques (Mar 1996)

Quantitative RT-PCR on CYP1A1 Heterogeneous Nuclear RNA: A Surrogate for the In Vitro Transcription Run-On Assay

  • Cornelis J. Elferink,
  • John J. Reiners

DOI
https://doi.org/10.2144/19962003470
Journal volume & issue
Vol. 20, no. 3
pp. 470 – 477

Abstract

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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 introndirected 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 Cypla-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 Cypla-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.