Determination of murine fetal Cyp1a1 and 1b1 expression by real-time fluorescence reverse transcription-polymerase chain reaction

Abstract

We describe the application of quantitative RT-PCR methodology to study expression of Cyp1a1 and 1b1 in murine fetal tissue. We applied real-time PCR using the fluorescent dye, SYBR Green, to establish a very reliable, sensitive, specific, inexpensive, and rapid assay that was linear over a wide dynamic range of starting target gene copy number. The main point of this paper is to provide a detailed description of how to optimize real-time reverse transcription-polymerase chain reaction (RT-PCR) assays and, most importantly, to provide solutions for some of the commonly observed problems encountered when employing quantitative RT-PCR methods. We establish that use of SYBR Green for detection of PCR products was a powerful and reliable tool in our investigation of Cyp gene expression in murine fetal tissues, and demonstrate the kinetics of induction of Cyp1a1 and 1b1 in crosses between C57BL/6 and Balb/c mice. In addition, we demonstrate for the first time the induction of Cyp1b1 RNA expression in fetal lung and liver tissues following in utero exposure to 3-methylcholanthrene (MC), a polycyclic aromatic hydrocarbon. This assay for Cyp1a1 and 1b1 expression could be utilized equally well to detect other genes of interest in a variety of toxicological investigations.