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

Cornelis J. Elferink, John J. Reiners

Research output: Contribution to journalArticle

134 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)470-477
Number of pages8
JournalBioTechniques
Volume20
Issue number3
DOIs
StatePublished - Mar 1996

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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