QUANTITATIVE RT-PCR ON CYP1A1 HETEROGENEOUS NUCLEAR-RNA - A SURROGATEFOR THE IN-VITRO TRANSCRIPTION RUN-ON ASSAY

A quantitative reverse se transcription polymerase chain reaction (RT- PCR) assay was developed to amplify a region of the CYP1A1 heterogeneo us nuclear RNA (hnRNA) transcript encompassing the first intron-exon b oundary. The RT-PCR protocol uses a CYP1A1 recombinant RNA internal st andard identical to the ml get hnRNA except for an engineered unique i nternal restriction site. Its inclusion enables normalization between reactions and a measurement of the absolute number of target hnRNA tra nscripts. Specificity for the hnRNA was achieved by using intron-direc ted primers in both the RT and the PCR. Nuclear run-on assays and the hnRNA RT-PCR using detected an equivalent increase in transcription of Cyp1a-1 in cultured murine Hepa 1c1 c7 cells following exposure to 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The RT-PCR assay also reveal ed TCDD-dependent transcriptional activation of the Cyp1a-1 gene in mu rine skin, a tissue unsuited to the nuclear run-on assay because of in herent difficulties associated with the isolation of nuclei. These exa mples demonstrate that the hnRNA RT-PCR assay is a facile surrogate fo r the nuclear run-on assay. Moreover; the sensitivity and design chara cteristics of the RT-PCR assay suggest the potential Soi its broad app lication in general transcriptional research.