Determination of murine fetal Cyp1a1 and 1b1 expression by real-time fluorescence reverse transcription-polymerase chain reaction.
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.
Xu
Mian
M
Department of Cancer Biology, Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1082, USA.
Miller
Mark Steven
MS
D000465:Algorithms; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D003199:Computer Systems; D019363:Cytochrome P-450 CYP1A1 / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D065633:Cytochrome P-450 CYP1B1; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D005333:Fetus / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D008099:Liver / Q000196:embryology / Q000201:enzymology; D008168:Lung / Q000196:embryology / Q000201:enzymology; D008748:Methylcholanthrene / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D011247:Pregnancy; D011786:Quality Control; D012313:RNA / Q000096:biosynthesis; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D018076:DNA, Complementary; D008748:Methylcholanthrene; D012313:RNA; D001189:Aryl Hydrocarbon Hydroxylases; C585498:Cyp1b1 protein, mouse; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1